U.S. patent number 10,683,351 [Application Number 16/291,826] was granted by the patent office on 2020-06-16 for antibody constructs for dll3 and cd3.
This patent grant is currently assigned to AMGEN RESEARCH (MUNICH) GMBH. The grantee listed for this patent is AMGEN RESEARCH (MUNICH) GMBH. Invention is credited to Claudia Blumel, Christoph Dahlhoff, Patrick Hoffmann, Peter Kufer, Ralf Lutterbuse, Elisabeth Nahrwold, Jochen Pendzialek, Tobias Raum.
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United States Patent |
10,683,351 |
Raum , et al. |
June 16, 2020 |
Antibody constructs for DLL3 and CD3
Abstract
The present invention relates to a bispecific antibody construct
comprising a first binding domain which binds to human DLL3 on the
surface of a target cell and a second binding domain which binds to
human CD3 on the surface of a T cell. Moreover, the invention
provides a polynucleotide encoding the antibody construct, a vector
comprising the polynucleotide and a host cell transformed or
transfected with the polynucleotide or vector. Furthermore, the
invention provides a process for the production of the antibody
construct of the invention, a medical use of the antibody construct
and a kit comprising the antibody construct.
Inventors: |
Raum; Tobias (Munich,
DE), Blumel; Claudia (Munich, DE),
Dahlhoff; Christoph (Munich, DE), Hoffmann;
Patrick (Munich, DE), Kufer; Peter (Munich,
DE), Lutterbuse; Ralf (Munich, DE),
Nahrwold; Elisabeth (Munich, DE), Pendzialek;
Jochen (Munich, DE) |
Applicant: |
Name |
City |
State |
Country |
Type |
AMGEN RESEARCH (MUNICH) GMBH |
Munich |
N/A |
DE |
|
|
Assignee: |
AMGEN RESEARCH (MUNICH) GMBH
(Munich, DE)
|
Family
ID: |
56800252 |
Appl.
No.: |
16/291,826 |
Filed: |
March 4, 2019 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20190263907 A1 |
Aug 29, 2019 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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15225107 |
Aug 1, 2016 |
10294300 |
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62290896 |
Feb 3, 2016 |
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62199930 |
Jul 31, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K
39/3955 (20130101); A61P 35/00 (20180101); C12N
15/63 (20130101); A61K 39/00113 (20180801); C07K
16/2809 (20130101); A61K 39/39558 (20130101); C07K
16/28 (20130101); C07K 16/18 (20130101); C12N
5/06 (20130101); C07K 2317/515 (20130101); C07K
2317/56 (20130101); C07K 16/22 (20130101); C07K
2317/622 (20130101); C07K 2317/34 (20130101); C07K
2317/76 (20130101); A61K 2039/505 (20130101); C07K
2317/73 (20130101); C07K 2317/94 (20130101); C07K
2317/31 (20130101); C07K 2317/51 (20130101); C07K
2317/77 (20130101); C07K 2317/92 (20130101); C07K
16/30 (20130101); C07K 2317/33 (20130101) |
Current International
Class: |
A61K
39/395 (20060101); C07K 16/18 (20060101); A61K
39/00 (20060101); C07K 16/22 (20060101); C07K
16/28 (20060101); C07K 16/30 (20060101); C12N
5/07 (20100101); C12N 15/63 (20060101); C07K
14/475 (20060101); C07K 14/725 (20060101) |
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Primary Examiner: Bunner; Bridget E
Attorney, Agent or Firm: Marshall, Gerstein & Borun
LLP
Claims
The invention claimed is:
1. A nucleic acid encoding a bispecific antibody construct
comprising a first binding domain which binds to human delta-like 3
(DLL3) on the surface of a target cell and a second binding domain
which binds to human and macaque CD3 on the surface of a T cell,
wherein the first binding domain binds to an epitope of DLL3 within
the amino acid sequence of SEQ ID NO: 258.
2. The nucleic acid of claim 1, wherein the first binding domain
further binds to macaque DLL3.
3. The nucleic acid of claim 2, wherein the macaque DLL3 is Macaca
fascicularis DLL3.
4. The nucleic acid of claim 1, wherein the second binding domain
binds to human CD3 epsilon and to Callithrix jacchus, Saguinus
Oedipus or Saimiri sciureus CD3 epsilon.
5. The nucleic acid of claim 1, wherein the antibody construct is
in a format selected from the group consisting of: (scFv)2,
diabodies and oligomers of the foregoing formats.
6. The nucleic acid of claim 1, wherein the first binding domain
comprises a VH region comprising CDR-H1, CDR-H2 and CDR-H3 and a VL
region comprising CDR-L1, CDR-L2 and CDR-L3 selected from the group
consisting of: a) CDR-H1 comprising the amino acid sequence of SEQ
ID NO: 31, CDR-H2 comprising the amino acid sequence of SEQ ID NO:
32, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 33,
CDR-L1 comprising the amino acid sequence of SEQ ID NO: 34, CDR-L2
comprising the amino acid sequence of SEQ ID NO: 35, and CDR-L3
comprising the amino acid sequence of SEQ ID NO: 36; b) CDR-H1
comprising the amino acid sequence of SEQ ID NO: 41, CDR-H2
comprising the amino acid sequence of SEQ ID NO: 42, CDR-H3
comprising the amino acid sequence of SEQ ID NO: 43, CDR-L1
comprising the amino acid sequence of SEQ ID NO: 44, CDR-L2
comprising the amino acid sequence of SEQ ID NO: 45, and CDR-L3
comprising the amino acid sequence of SEQ ID NO: 46; c) CDR-H1
comprising the amino acid sequence of SEQ ID NO: 51, CDR-H2
comprising the amino acid sequence of SEQ ID NO: 52, CDR-H3
comprising the amino acid sequence of SEQ ID NO: 53, CDR-L1
comprising the amino acid sequence of SEQ ID NO: 54, CDR-L2
comprising the amino acid sequence of SEQ ID NO: 55, and CDR-L3
comprising the amino acid sequence of SEQ ID NO: 56; d) CDR-H1
comprising the amino acid sequence of SEQ ID NO: 61, CDR-H2
comprising the amino acid sequence of SEQ ID NO: 62, CDR-H3
comprising the amino acid sequence of SEQ ID NO: 63, CDR-L1
comprising the amino acid sequence of SEQ ID NO: 64, CDR-L2
comprising the amino acid sequence of SEQ ID NO: 65, and CDR-L3
comprising the amino acid sequence of SEQ ID NO: 66; e) CDR-H1
comprising the amino acid sequence of SEQ ID NO: 71, CDR-H2
comprising the amino acid sequence of SEQ ID NO: 72, CDR-H3
comprising the amino acid sequence of SEQ ID NO: 73, CDR-L1
comprising the amino acid sequence of SEQ ID NO: 74, CDR-L2
comprising the amino acid sequence of SEQ ID NO: 75, and CDR-L3
comprising the amino acid sequence of SEQ ID NO: 76; f) CDR-H1
comprising the amino acid sequence of SEQ ID NO: 81, CDR-H2
comprising the amino acid sequence of SEQ ID NO: 82, CDR-H3
comprising the amino acid sequence of SEQ ID NO: 83, CDR-L1
comprising the amino acid sequence of SEQ ID NO: 84, CDR-L2
comprising the amino acid sequence of SEQ ID NO: 85, and CDR-L3
comprising the amino acid sequence of SEQ ID NO: 86; g) CDR-H1
comprising the amino acid sequence of SEQ ID NO: 91, CDR-H2
comprising the amino acid sequence of SEQ ID NO: 92, CDR-H3
comprising the amino acid sequence of SEQ ID NO: 93, CDR-L1
comprising the amino acid sequence of SEQ ID NO: 94, CDR-L2
comprising the amino acid sequence of SEQ ID NO: 95, and CDR-L3
comprising the amino acid sequence of SEQ ID NO: 96; h) CDR-H1
comprising the amino acid sequence of SEQ ID NO: 101, CDR-H2
comprising the amino acid sequence of SEQ ID NO: 102, CDR-H3
comprising the amino acid sequence of SEQ ID NO: 103, CDR-L1
comprising the amino acid sequence of SEQ ID NO: 104, CDR-L2
comprising the amino acid sequence of SEQ ID NO: 105, and CDR-L3
comprising the amino acid sequence of SEQ ID NO: 106; and i) CDR-H1
comprising the amino acid sequence of SEQ ID NO: 111, CDR-H2
comprising the amino acid sequence of SEQ ID NO: 112, CDR-H3
comprising the amino acid sequence of SEQ ID NO: 113, CDR-L1
comprising the amino acid sequence of SEQ ID NO: 114, CDR-L2
comprising the amino acid sequence of SEQ ID NO: 115, and CDR-L3
comprising the amino acid sequence of SEQ ID NO: 116.
7. The nucleic acid of claim 6, wherein the second binding domain
comprises a VL region comprising CDR-H1, CDR-H2 and CDR-H3 and a VH
region comprising CDR-L1, CDR-L2 and CDR-L3 selected from the group
consisting of: a) CDR-L1 comprising the amino acid sequence of SEQ
ID NO: 342, CDR-L2 comprising the amino acid sequence of SEQ ID NO:
343, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 344;
CDR-H1 comprising the amino acid sequence of SEQ ID NO: 345, CDR-H2
comprising the amino acid sequence of SEQ ID NO: 346, and CDR-H3
comprising the amino acid sequence of SEQ ID NO: 347; b) CDR-L1
comprising the amino acid sequence of SEQ ID NO: 351, CDR-L2
comprising the amino acid sequence of SEQ ID NO: 352, CDR-L3
comprising the amino acid sequence of SEQ ID NO: 353; CDR-H1
comprising the amino acid sequence of SEQ ID NO: 354, CDR-H2
comprising the amino acid sequence of SEQ ID NO: 355, and CDR-H3
comprising the amino acid sequence of SEQ ID NO: 356; c) CDR-L1
comprising the amino acid sequence of SEQ ID NO: 360, CDR-L2
comprising the amino acid sequence of SEQ ID NO: 361, CDR-L3
comprising the amino acid sequence of SEQ ID NO: 362; CDR-H1
comprising the amino acid sequence of SEQ ID NO: 363, CDR-H2
comprising the amino acid sequence of SEQ ID NO: 364, and CDR-H3
comprising the amino acid sequence of SEQ ID NO: 365; d) CDR-L1
comprising the amino acid sequence of SEQ ID NO: 369, CDR-L2
comprising the amino acid sequence of SEQ ID NO: 370, CDR-L3
comprising the amino acid sequence of SEQ ID NO: 371; CDR-H1
comprising the amino acid sequence of SEQ ID NO: 372, CDR-H2
comprising the amino acid sequence of SEQ ID NO: 373, and CDR-H3
comprising the amino acid sequence of SEQ ID NO: 374; e) CDR-L1
comprising the amino acid sequence of SEQ ID NO: 378, CDR-L2
comprising the amino acid sequence of SEQ ID NO: 379, CDR-L3
comprising the amino acid sequence of SEQ ID NO: 380; CDR-H1
comprising the amino acid sequence of SEQ ID NO: 381, CDR-H2
comprising the amino acid sequence of SEQ ID NO: 382, and CDR-H3
comprising the amino acid sequence of SEQ ID NO: 383; f) CDR-L1
comprising the amino acid sequence of SEQ ID NO: 387, CDR-L2
comprising the amino acid sequence of SEQ ID NO: 388, CDR-L3
comprising the amino acid sequence of SEQ ID NO: 389; CDR-H1
comprising the amino acid sequence of SEQ ID NO: 390, CDR-H2
comprising the amino acid sequence of SEQ ID NO: 391, and CDR-H3
comprising the amino acid sequence of SEQ ID NO: 392; g) CDR-L1
comprising the amino acid sequence of SEQ ID NO: 396, CDR-L2
comprising the amino acid sequence of SEQ ID NO: 397, CDR-L3
comprising the amino acid sequence of SEQ ID NO: 398; CDR-H1
comprising the amino acid sequence of SEQ ID NO: 399, CDR-H2
comprising the amino acid sequence of SEQ ID NO: 400, and CDR-H3
comprising the amino acid sequence of SEQ ID NO: 401; h) CDR-L1
comprising the amino acid sequence of SEQ ID NO: 405, CDR-L2
comprising the amino acid sequence of SEQ ID NO: 406, CDR-L3
comprising the amino acid sequence of SEQ ID NO: 407; CDR-H1
comprising the amino acid sequence of SEQ ID NO: 408, CDR-H2
comprising the amino acid sequence of SEQ ID NO: 409, and CDR-H3
comprising the amino acid sequence of SEQ ID NO: 410; and i) CDR-L1
comprising the amino acid sequence of SEQ ID NO: 414, CDR-L2
comprising the amino acid sequence of SEQ ID NO: 415, CDR-L3
comprising the amino acid sequence of SEQ ID NO: 416; CDR-H1
comprising the amino acid sequence of SEQ ID NO: 417, CDR-H2
comprising the amino acid sequence of SEQ ID NO: 418, and CDR-H3
comprising the amino acid sequence of SEQ ID NO: 419; and j) CDR-L1
comprising the amino acid sequence of SEQ ID NO: 423, CDR-L2
comprising the amino acid sequence of SEQ ID NO: 424, CDR-L3
comprising the amino acid sequence of SEQ ID NO: 425; CDR-H1
comprising the amino acid sequence of SEQ ID NO: 426, CDR-H2
comprising the amino acid sequence of SEQ ID NO: 427, and CDR-H3
comprising the amino acid sequence of SEQ ID NO: 428.
8. The nucleic acid of claim 6, wherein the second binding domain
comprises a VH region comprising the amino acid sequence selected
from the group consisting of: SEQ ID NO: 348, SEQ ID NO: 357, SEQ
ID NO: 366, SEQ ID NO: 375, SEQ ID NO: 384, SEQ ID NO: 393, SEQ ID
NO: 402, SEQ ID NO: 411, SEQ ID NO: 420, and SEQ ID NO: 429.
9. The nucleic acid of claim 6, wherein the second binding domain
comprises a VL region comprising the amino acid sequence selected
from the group consisting of: SEQ ID NO: 349, SEQ ID NO: 358, SEQ
ID NO: 367, SEQ ID NO: 376, SEQ ID NO: 385, SEQ ID NO: 394, SEQ ID
NO: 403, SEQ ID NO: 412, SEQ ID NO: 421, and SEQ ID NO: 430.
10. The nucleic acid of claim 6, wherein the second binding domain
comprises a VH region and a VL region comprising the pair of amino
acid sequences, respectively, selected from the group consisting
of: SEQ ID NOs: 348 and 349; SEQ ID NOs: 357 and 358; SEQ ID NOs:
366 and 367; SEQ ID NOs: 375 and 376; SEQ ID NOs: 384 and 385; SEQ
ID NOs: 393 and 394; SEQ ID NOs: 402 and 403; SEQ ID NOs: 411 and
412; SEQ ID NOs: 420 and 421; and SEQ ID NOs: 429 and 430.
11. The nucleic acid of claim 6, wherein the second binding domain
comprises an amino acid sequence selected from the group consisting
of: SEQ ID NO: 350, SEQ ID NO: 359, SEQ ID NO: 368, SEQ ID NO: 377,
SEQ ID NO: 386, SEQ ID NO: 395, SEQ ID NO: 404, SEQ ID NO: 413, SEQ
ID NO: 422, and SEQ ID NO: 431.
12. The nucleic acid of claim 6, wherein the first binding domain
comprises a VH region comprising a CDR-H1 comprising the amino acid
sequence of SEQ ID NO: 31, a CDR-H2 comprising the amino acid
sequence of SEQ ID NO: 32, and a CDR-H3 comprising the amino acid
sequence of SEQ ID NO: 33, and a VL region comprising a CDR-L1
comprising the amino acid sequence of SEQ ID NO: 34, a CDR-L2
comprising the amino acid sequence of SEQ ID NO: 35, and a CDR-L3
comprising the amino acid sequence of SEQ ID NO: 36, and wherein
the second binding domain comprises a VL domain comprising a CDR-L1
comprising the amino acid sequence of SEQ ID NO: 423, a CDR-L2
comprising the amino acid sequence of SEQ ID NO: 424, a CDR-L3
comprising the amino acid sequence of SEQ ID NO: 425, and a VH
domain comprising a CDR-H1 comprising the amino acid sequence of
SEQ ID NO: 426, a CDR-H2 comprising the amino acid sequence of SEQ
ID NO: 427, and a CDR-H3 comprising the amino acid sequence of SEQ
ID NO: 428.
13. The nucleic acid of claim 12, wherein the first binding domain
comprises a VH region comprising the amino acid sequence of SEQ ID
NO: 435 and a VL region comprising the amino acid sequence of SEQ
ID NO: 436, and wherein the second binding domain comprises a VH
region comprising the amino acid sequence of SEQ ID NO: 429 and a
VL region comprising the amino acid sequence of SEQ ID NO: 430.
14. The nucleic acid of claim 13, wherein the first binding domain
comprises the amino acid sequence of SEQ ID NO: 437, and wherein
the second binding domain comprises the amino acid sequence of SEQ
ID NO: 431.
15. The nucleic acid of claim 1, wherein the first binding domain
comprises a VH region comprising the amino acid sequence selected
from the group consisting of: SEQ ID NO: 37, SEQ ID NO: 47, SEQ ID
NO: 57, SEQ ID NO: 67, SEQ ID NO: 77, SEQ ID NO: 87, SEQ ID NO: 97,
SEQ ID NO: 107, SEQ ID NO: 117, SEQ ID NO: 435, and SEQ ID NO:
529.
16. The nucleic acid of claim 1, wherein the first binding domain
comprises a VL region comprising the amino acid sequence selected
from the group consisting of: SEQ ID NO: 38, SEQ ID NO: 48, SEQ ID
NO: 58, SEQ ID NO: 68, SEQ ID NO: 78, SEQ ID NO: 88, SEQ ID NO: 98,
SEQ ID NO: 108, SEQ ID NO: 118, SEQ ID NO: 436, and SEQ ID NO:
530.
17. The nucleic acid of claim 1, wherein the first binding domain
comprises a VH region and a VL region comprising the pair of amino
acid sequences, respectively, selected from the group consisting
of: SEQ ID NOs: 37+38; SEQ ID NOs: 47+48; SEQ ID NOs: 57+58; SEQ ID
NOs: 67+68; SEQ ID NOs: 77+78; SEQ ID NOs: 87+88; SEQ ID NOs:
97+98; SEQ ID NOs: 107+108; SEQ ID NOs: 117+118; SEQ ID NOs:
435+436; and SEQ ID NOs: 529+530.
18. The nucleic acid of claim 1, wherein the first binding domain
comprises a polypeptide comprising the amino acid sequence selected
from the group consisting of: SEQ ID NO: 39, SEQ ID NO: 49, SEQ ID
NO: 59, SEQ ID NO: 69, SEQ ID NO: 79, SEQ ID NO: 89, SEQ ID NO: 99,
SEQ ID NO: 109, SEQ ID NO: 119, SEQ ID NO: 437, and SEQ ID NO:
531.
19. The nucleic acid of claim 1, wherein the nucleic acid comprises
a nucleotide sequence encoding a polypeptide comprising the amino
acid sequence selected from the group consisting of: SEQ ID NO: 40,
SEQ ID NO: 50, SEQ ID NO: 60, SEQ ID NO: 70, SEQ ID NO: 80, SEQ ID
NO: 90, SEQ ID NO: 100, SEQ ID NO: 110, SEQ ID NO: 120, SEQ ID NO:
211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO:
215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 438, and SEQ ID NO:
532.
20. The nucleic acid of claim 1, wherein the nucleic acid comprises
a nucleotide sequence encoding a polypeptide comprising the amino
acid sequence selected from the group consisting of: SEQ ID NO:
517, SEQ ID NO: 518, SEQ ID NO: 519, SEQ ID NO: 520, SEQ ID NO:
521, SEQ ID NO: 522, SEQ ID NO: 523, and SEQ ID NO: 524.
21. A vector comprising the nucleic acid of claim 1.
22. An isolated host cell transformed or transfected with the
vector of claim 21.
23. A process for producing a bispecific antibody construct
comprising culturing the host cell of claim 22 under conditions
allowing the expression of the antibody construct and, optionally,
recovering the antibody construct from the culture.
24. An isolated host cell transformed or transfected with the
nucleic acid of claim 1.
Description
The present invention relates to a bispecific antibody construct
comprising a first binding domain which binds to human DLL3 on the
surface of a target cell and a second binding domain which binds to
human CD3 on the surface of a T cell. Moreover, the invention
provides a polynucleotide encoding the antibody construct, a vector
comprising said polynucleotide and a host cell transformed or
transfected with said polynucleotide or vector. Furthermore, the
invention provides a process for the production of the antibody
construct of the invention, a medical use of said antibody
construct and a kit comprising said antibody construct.
Small cell lung cancer (SCLC) is an aggressive form of lung cancer
with a poor prognosis and limited therapeutic options, representing
about 15% of all newly diagnosed lung cancers and equal to about
25,000 new cases in the US and 180,000 new cases worldwide per
year. Survival rates have remained low for several decades, with
only 5% of SCLC patients surviving five years, in a large part due
to the lack of new therapies to combat this form of lung cancer.
Most patients present with extensive-stage disease, while about a
third of patients present with limited stage disease, defined by
the presence of tumors in only one side of the chest and that fit
in a single radiation field. These stages impact available
therapeutic regiments, with limited stage disease treated with
chemotherapy and radiation and extensive stage disease treated with
chemotherapy alone. Disseminated, metastatic tumors with
lymphoma-like characteristics are a hallmark of SCLC. The first
known diagnosis of SCLC patients described it as a disease of the
lymphatic system, not being recognized as lung cancer until 1926,
which highlights some of the unique nature of SCLC tumors as
compared to other solid tumors.
Patients typically respond well to the current front-line therapy,
which includes etoposide and cisplatin, but invariably quickly
relapse with chemoresistant disease, for which no therapeutic
options are currently available. Prognosis in the relapsed
refractory setting is extremely poor, with rapid disease
progression and short median survival of less than six months.
Furthermore, SCLC patients have high rates of comorbidities,
including hypertension, cardiac disease, diabetes and
paraneoplastic syndromes. These, coupled with the typically
advanced age of SCLC patients, impact the ability of patients to
endure harsh chemo regimens, further limiting treatment
options.
A bispecific antibody modality, comprising an scFv that recognizes
CD3 expressed on T cells and another scFv that recognizes a
tumor-associated antigen, has shown promising efficacy in the
clinic, with high response rates in hematological malignancies such
as refractory B-ALL (Topp, M. S. et al. Blood, 2012. 120(26): p.
5185-5187), resulting in the approval of Blincyto. While efficacy
with T cell-engaging therapies has yet to be demonstrated in a
solid tumor indication, SCLC may represent a promising solid tumor
indication for the CD3.times. tumor target bispecific antibody
modality, given the disseminated nature of the disease. Therefore,
a bispecific T cell engager that directs T cells against a specific
tumor antigen presents a new opportunity as a new therapeutic
option in the treatment of SCLC.
DLL3 was presently identified as an SCLC-specific tumor antigen by
next-generation sequencing, comparing the prevalence of DLL3 mRNA
in a panel of primary patient tumors and a large collection of
normal tissues. The level of DLL3 expression in SCLC tumors was
moderate, but highly prevalent, with approximately 90% of the
tumors analyzed showing evidence of DLL3 expression by RNA-seq. In
contrast to SCLC tumors, normal tissues showed very low expression
of DLL3 transcript, with small levels detected in testis, optic
nerve and cerebellum. Comparison of SCLC cell lines and tumors by
RNA-seq showed similar expression levels, while cell surface
quantitation of DLL3 expression on SCLC cell lines indicated
expression levels below 5000 DLL3 per cell, with typical expression
levels below 2000 DLL3/cell. Expression of DLL3 protein was
confirmed by IHC, where 86% of SCLC tumors showed positive staining
for DLL3, with a homogeneous and membranous staining pattern. Aside
from very faint staining in cerebellum, all other normal tissues
were negative for DLL3 staining.
DLL3 is a non-canonical Notch ligand, functioning in a cell
autonomous manner to inhibit Notch signaling, binding to Notch in
cis, thus blocking cell to cell interactions and internalization of
Notch in the target cell, a hallmark of canonical Notch signaling.
The primary role for DLL3 is in somitogenesis during embryonic
development. Mice with DLL3 knockouts show segmental defects in the
axial skeleton and cranial and neuronal development. Somitic
patterning defects are also seen in humans with certain germline
DLL3 mutations, resulting in a condition called spondylocostal
dysostosis.
DLL3 has been proposed previously in methods to diagnose and treat
glioma, in addition to SCLC, using an antibody-drug conjugate (ADC)
(WO 2013/126746). Using an ADC-based approach for DLL3 may have
limitations, given the low expression levels of the protein on the
cell surface and the reduced performance of ADCs against targets
with low expression. Furthermore, ADC molecules often demonstrate
toxicity related to free warhead, likely a result of linker
degradation, resulting in maximum tolerated dose limitations and
potential impacts on efficacy unrelated to the target chosen for
the antibody. This is less likely to be an issue for a T
cell-engaging bispecific molecule, engineered to engage DLL3 and
CD3 simultaneously, given the required sensitivity of T cells for
their targets, and highly potent in vitro cytotoxicity has been
demonstrated on cell lines expressing several hundred target
proteins per cell. Additionally, the usually smaller size of a
bispecific T cell engaging antibody construct relative to a normal
antibody (full-length IgG) may improve tissue penetration and
increase potency due to more efficient engagement of the DLL3 and
CD3 targets, resulting in improved synapse formation between the T
cell and target tumor cell.
SCLC remains a significant unmet medical need, and new therapeutic
options are required to improve the outlook for this sizable
patient population. The above discussed bispecific antibody
modality is clinically validated, and as such an antibody construct
targeting DLL3 and CD3 represents a promising new possibility for
the treatment of SCLC and an opportunity to improve the survival of
patients suffering with this indication. As there is still a need
for having available further options for the treatment of tumor or
cancer diseases related to the overexpression of DLL3, there are
provided herewith means and methods for the solution of this
problem in the form of a bispecific antibody construct with one
binding domain directed to DLL3 and a second binding domain
directed to CD3 on T cells.
Thus, in a first aspect, the present invention provides a
bispecific antibody construct comprising a first binding domain
which binds to human DLL3 on the surface of a target cell and a
second binding domain which binds to human CD3 on the surface of a
T cell, wherein the first binding domain binds to an epitope of
DLL3 which is comprised within the region as depicted in SEQ ID NO:
260.
It must be noted that as used herein, the singular forms "a", "an",
and "the" include plural references unless the context clearly
indicates otherwise. Thus, for example, reference to "a reagent"
includes one or more of such different reagents and reference to
"the method" includes reference to equivalent steps and methods
known to those of ordinary skill in the art that could be modified
or substituted for the methods described herein.
Unless otherwise indicated, the term "at least" preceding a series
of elements is to be understood to refer to every element in the
series. Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
present invention.
The term "and/or" wherever used herein includes the meaning of
"and", "or" and "all or any other combination of the elements
connected by said term".
The term "about" or "approximately" as used herein means within
.+-.20%, preferably within .+-.15%, more preferably within .+-.10%,
and most preferably within .+-.5% of a given value or range.
Throughout this specification and the claims which follow, unless
the context requires otherwise, the word "comprise", and variations
such as "comprises" and "comprising", will be understood to imply
the inclusion of a stated integer or step or group of integers or
steps but not the exclusion of any other integer or step or group
of integer or step. When used herein the term "comprising" can be
substituted with the term "containing" or "including" or sometimes
when used herein with the term "having".
When used herein "consisting of" excludes any element, step, or
ingredient not specified in the claim element. When used herein,
"consisting essentially of" does not exclude materials or steps
that do not materially affect the basic and novel characteristics
of the claim.
In each instance herein any of the terms "comprising", "consisting
essentially of" and "consisting of" may be replaced with either of
the other two terms.
The term "antibody construct" refers to a molecule in which the
structure and/or function is/are based on the structure and/or
function of an antibody, e.g., of a full-length or whole
immunoglobulin molecule. An antibody construct is hence capable of
binding to its specific target or antigen. Furthermore, an antibody
construct according to the invention comprises the minimum
structural requirements of an antibody which allow for the target
binding. This minimum requirement may e.g. be defined by the
presence of at least the three light chain CDRs (i.e. CDR1, CDR2
and CDR3 of the VL region) and/or the three heavy chain CDRs (i.e.
CDR1, CDR2 and CDR3 of the VH region), preferably of all six CDRs.
The antibodies on which the constructs according to the invention
are based include for example monoclonal, recombinant, chimeric,
deimmunized, humanized and human antibodies.
Within the definition of "antibody constructs" according to the
invention are full-length or whole antibodies also including
camelid antibodies and other immunoglobulin antibodies generated by
biotechnological or protein engineering methods or processes. These
full-length antibodies may be for example monoclonal, recombinant,
chimeric, deimmunized, humanized and human antibodies. Also within
the definition of "antibody constructs" are fragments of
full-length antibodies, such as VH, VHH, VL, (s)dAb, Fv, Fd, Fab,
Fab', F(ab')2 or "r IgG" ("half antibody"). Antibody constructs
according to the invention may also be modified fragments of
antibodies, also called antibody variants, such as scFv, di-scFv or
bi(s)-scFv, scFv-Fc, scFv-zipper, scFab, Fab2, Fab3, diabodies,
single chain diabodies, tandem diabodies (Tandab's), tandem
di-scFv, tandem tri-scFv, "minibodies" exemplified by a structure
which is as follows: (VH-VL-CH3)2, (scFv-CH3)2, ((scFv)2-CH3+CH3),
((scFv)2-CH3) or (scFv-CH3-scFv)2, multibodies such as triabodies
or tetrabodies, and single domain antibodies such as nanobodies or
single variable domain antibodies comprising merely one variable
domain, which might be VHH, VH or VL, that specifically bind an
antigen or epitope independently of other V regions or domains.
Further preferred formats of the antibody constructs according to
the invention are cross bodies, maxi bodies, hetero Fc constructs
and mono Fc constructs. Examples for those formats will be
described herein below.
A binding domain may typically comprise an antibody light chain
variable region (VL) and an antibody heavy chain variable region
(VH); however, it does not have to comprise both. Fd fragments, for
example, have two VH regions and often retain some antigen-binding
function of the intact antigen-binding domain. Additional examples
for the format of antibody fragments, antibody variants or binding
domains include (1) a Fab fragment, a monovalent fragment having
the VL, VH, CL and CH1 domains; (2) a F(ab')2 fragment, a bivalent
fragment having two Fab fragments linked by a disulfide bridge at
the hinge region; (3) an Fd fragment having the two VH and CH1
domains; (4) an Fv fragment having the VL and VH domains of a
single arm of an antibody, (5) a dAb fragment (Ward et al., (1989)
Nature 341:544-546), which has a VH domain; (6) an isolated
complementarity determining region (CDR), and (7) a single chain Fv
(scFv), the latter being preferred (for example, derived from an
scFv-library). Examples for embodiments of antibody constructs
according to the invention are e.g. described in WO 00/006605, WO
2005/040220, WO 2008/119567, WO 2010/037838, WO 2013/026837, WO
2013/026833, US 2014/0308285, US 2014/0302037, WO 2014/144722, WO
2014/151910, and WO 2015/048272.
Furthermore, the definition of the term "antibody construct"
includes monovalent, bivalent and polyvalent/multivalent constructs
and, thus, monospecific constructs, specifically binding to only
one antigenic structure, as well as bispecific and
polyspecific/multispecific constructs, which specifically bind more
than one antigenic structure, e.g. two, three or more, through
distinct binding domains. Moreover, the definition of the term
"antibody construct" includes molecules consisting of only one
polypeptide chain as well as molecules consisting of more than one
polypeptide chain, which chains can be either identical
(homodimers, homotrimers or homo oligomers) or different
(heterodimer, heterotrimer or heterooligomer). Examples for the
above identified antibodies and variants or derivatives thereof are
described inter alia in Harlow and Lane, Antibodies a laboratory
manual, CSHL Press (1988) and Using Antibodies: a laboratory
manual, CSHL Press (1999), Kontermann and Dubel, Antibody
Engineering, Springer, 2nd ed. 2010 and Little, Recombinant
Antibodies for Immunotherapy, Cambridge University Press 2009.
The antibody constructs of the present invention are preferably "in
vitro generated antibody constructs". This term refers to an
antibody construct according to the above definition where all or
part of the variable region (e.g., at least one CDR) is generated
in a non-immune cell selection, e.g., an in vitro phage display,
protein chip or any other method in which candidate sequences can
be tested for their ability to bind to an antigen. This term thus
preferably excludes sequences generated solely by genomic
rearrangement in an immune cell in an animal. A "recombinant
antibody" is an antibody made through the use of recombinant DNA
technology or genetic engineering.
The term "monoclonal antibody" (mAb) or monoclonal antibody
construct as used herein refers to an antibody obtained from a
population of substantially homogeneous antibodies, i.e., the
individual antibodies comprising the population are identical
except for possible naturally occurring mutations and/or
post-translation modifications (e.g., isomerizations, amidations)
that may be present in minor amounts. Monoclonal antibodies are
highly specific, being directed against a single antigenic site or
determinant on the antigen, in contrast to conventional
(polyclonal) antibody preparations which typically include
different antibodies directed against different determinants (or
epitopes). In addition to their specificity, the monoclonal
antibodies are advantageous in that they are synthesized by the
hybridoma culture, hence uncontaminated by other immunoglobulins.
The modifier "monoclonal" indicates the character of the antibody
as being obtained from a substantially homogeneous population of
antibodies, and is not to be construed as requiring production of
the antibody by any particular method.
For the preparation of monoclonal antibodies, any technique
providing antibodies produced by continuous cell line cultures can
be used. For example, monoclonal antibodies to be used may be made
by the hybridoma method first described by Koehler et al., Nature,
256: 495 (1975), or may be made by recombinant DNA methods (see,
e.g., U.S. Pat. No. 4,816,567). Examples for further techniques to
produce human monoclonal antibodies include the trioma technique,
the human B-cell hybridoma technique (Kozbor, Immunology Today 4
(1983), 72) and the EBV-hybridoma technique (Cole et al.,
Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc.
(1985), 77-96).
Hybridomas can then be screened using standard methods, such as
enzyme-linked immunosorbent assay (ELISA) and surface plasmon
resonance (BIACORE.TM.) analysis, to identify one or more
hybridomas that produce an antibody that specifically binds with a
specified antigen. Any form of the relevant antigen may be used as
the immunogen, e.g., recombinant antigen, naturally occurring
forms, any variants or fragments thereof, as well as an antigenic
peptide thereof. Surface plasmon resonance as employed in the
BIAcore system can be used to increase the efficiency of phage
antibodies which bind to an epitope of a target antigen, such as
DLL3 or CD3 epsilon (Schier, Human Antibodies Hybridomas 7 (1996),
97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13).
Another exemplary method of making monoclonal antibodies includes
screening protein expression libraries, e.g., phage display or
ribosome display libraries. Phage display is described, for
example, in Ladner et al., U.S. Pat. No. 5,223,409; Smith (1985)
Science 228:1315-1317, Clackson et al., Nature, 352: 624-628 (1991)
and Marks et al., J. Mol. Biol., 222: 581-597 (1991).
In addition to the use of display libraries, the relevant antigen
can be used to immunize a non-human animal, e.g., a rodent (such as
a mouse, hamster, rabbit or rat). In one embodiment, the non-human
animal includes at least a part of a human immunoglobulin gene. For
example, it is possible to engineer mouse strains deficient in
mouse antibody production with large fragments of the human Ig
(immunoglobulin) loci. Using the hybridoma technology,
antigen-specific monoclonal antibodies derived from the genes with
the desired specificity may be produced and selected. See, e.g.,
XENOMOUSE.TM., Green et al. (1994) Nature Genetics 7:13-21, US
2003-0070185, WO 96/34096, and WO 96/33735.
A monoclonal antibody can also be obtained from a non-human animal,
and then modified, e.g., humanized, deimmunized, rendered chimeric
etc., using recombinant DNA techniques known in the art. Examples
of modified antibody constructs include humanized variants of
non-human antibodies, "affinity matured" antibodies (see, e.g.
Hawkins et al. J. Mol. Biol. 254, 889-896 (1992) and Lowman et al.,
Biochemistry 30, 10832-10837 (1991)) and antibody mutants with
altered effector function(s) (see, e.g., U.S. Pat. No. 5,648,260,
Kontermann and Dubel (2010), loc. cit. and Little (2009), loc.
cit.).
In immunology, affinity maturation is the process by which B cells
produce antibodies with increased affinity for antigen during the
course of an immune response. With repeated exposures to the same
antigen, a host will produce antibodies of successively greater
affinities. Like the natural prototype, the in vitro affinity
maturation is based on the principles of mutation and selection.
The in vitro affinity maturation has successfully been used to
optimize antibodies, antibody constructs, and antibody fragments.
Random mutations inside the CDRs are introduced using radiation,
chemical mutagens or error-prone PCR. In addition, the genetical
diversity can be increased by chain shuffling. Two or three rounds
of mutation and selection using display methods like phage display
usually results in antibody fragments with affinities in the low
nanomolar range.
A preferred type of an amino acid substitutional varianation of the
antibody constructs involves substituting one or more hypervariable
region residues of a parent antibody (e. g. a humanized or human
antibody). Generally, the resulting variant(s) selected for further
development will have improved biological properties relative to
the parent antibody from which they are generated. A convenient way
for generating such substitutional variants involves affinity
maturation using phage display. Briefly, several hypervariable
region sites (e. g. 6-7 sites) are mutated to generate all possible
amino acid substitutions at each site. The antibody variants thus
generated are displayed in a monovalent fashion from filamentous
phage particles as fusions to the gene III product of M13 packaged
within each particle. The phage-displayed variants are then
screened for their biological activity (e. g. binding affinity) as
herein disclosed. In order to identify candidate hypervariable
region sites for modification, alanine scanning mutagenesis can be
performed to identify hypervariable region residues contributing
significantly to antigen binding. Alternatively, or additionally,
it may be beneficial to analyze a crystal structure of the
antigen-antibody complex to identify contact points between the
binding domain and, e.g., human DLL3. Such contact residues and
neighbouring residues are candidates for substitution according to
the techniques elaborated herein. Once such variants are generated,
the panel of variants is subjected to screening as described herein
and antibodies with superior properties in one or more relevant
assays may be selected for further development.
The monoclonal antibodies and antibody constructs of the present
invention specifically include "chimeric" antibodies
(immunoglobulins) in which a portion of the heavy and/or light
chain is identical with or homologous to corresponding sequences in
antibodies derived from a particular species or belonging to a
particular antibody class or subclass, while the remainder of the
chain(s) is/are identical with or homologous to corresponding
sequences in antibodies derived from another species or belonging
to another antibody class or subclass, as well as fragments of such
antibodies, so long as they exhibit the desired biological activity
(U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci.
USA, 81: 6851-6855 (1984)). Chimeric antibodies of interest herein
include "primitized" antibodies comprising variable domain
antigen-binding sequences derived from a non-human primate (e.g.,
Old World Monkey, Ape etc.) and human constant region sequences. A
variety of approaches for making chimeric antibodies have been
described. See e.g., Morrison et al., Proc. Natl. Acad. ScL U.S.A.
81:6851, 1985; Takeda et al., Nature 314:452, 1985, Cabilly et al.,
U.S. Pat. No. 4,816,567; Boss et al., U.S. Pat. No. 4,816,397;
Tanaguchi et al., EP 0171496; EP 0173494; and GB 2177096.
An antibody, antibody construct, antibody fragment or antibody
variant may also be modified by specific deletion of human T cell
epitopes (a method called "deimmunization") by the methods
disclosed for example in WO 98/52976 or WO 00/34317. Briefly, the
heavy and light chain variable domains of an antibody can be
analyzed for peptides that bind to MHC class II; these peptides
represent potential T cell epitopes (as defined in WO 98/52976 and
WO 00/34317). For detection of potential T cell epitopes, a
computer modeling approach termed "peptide threading" can be
applied, and in addition a database of human MHC class II binding
peptides can be searched for motifs present in the VH and VL
sequences, as described in WO 98/52976 and WO 00/34317. These
motifs bind to any of the 18 major MHC class II DR allotypes, and
thus constitute potential T cell epitopes. Potential T cell
epitopes detected can be eliminated by substituting small numbers
of amino acid residues in the variable domains, or preferably, by
single amino acid substitutions. Typically, conservative
substitutions are made. Often, but not exclusively, an amino acid
common to a position in human germline antibody sequences may be
used. Human germline sequences are disclosed e.g. in Tomlinson, et
al. (1992) J. Mol. Biol. 227:776-798; Cook, G. P. et al. (1995)
Immunol. Today Vol. 16 (5): 237-242; and Tomlinson et al. (1995)
EMBO J. 14: 14:4628-4638. The V BASE directory provides a
comprehensive directory of human immunoglobulin variable region
sequences (compiled by Tomlinson, L A. et al. MRC Centre for
Protein Engineering, Cambridge, UK). These sequences can be used as
a source of human sequence, e.g., for framework regions and CDRs.
Consensus human framework regions can also be used, for example as
described in U.S. Pat. No. 6,300,064.
"Humanized" antibodies, antibody constructs, variants or fragments
thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding
subsequences of antibodies) are antibodies or immunoglobulins of
mostly human sequences, which contain (a) minimal sequence(s)
derived from non-human immunoglobulin. For the most part, humanized
antibodies are human immunoglobulins (recipient antibody) in which
residues from a hypervariable region (also CDR) of the recipient
are replaced by residues from a hypervariable region of a non-human
(e.g., rodent) species (donor antibody) such as mouse, rat, hamster
or rabbit having the desired specificity, affinity, and capacity.
In some instances, Fv framework region (FR) residues of the human
immunoglobulin are replaced by corresponding non-human residues.
Furthermore, "humanized antibodies" as used herein may also
comprise residues which are found neither in the recipient antibody
nor the donor antibody. These modifications are made to further
refine and optimize antibody performance. The humanized antibody
may also comprise at least a portion of an immunoglobulin constant
region (Fc), typically that of a human immunoglobulin. For further
details, see Jones et al., Nature, 321: 522-525 (1986); Reichmann
et al., Nature, 332: 323-329 (1988); and Presta, Curr. Op. Struct.
Biol., 2: 593-596 (1992).
Humanized antibodies or fragments thereof can be generated by
replacing sequences of the Fv variable domain that are not directly
involved in antigen binding with equivalent sequences from human Fv
variable domains. Exemplary methods for generating humanized
antibodies or fragments thereof are provided by Morrison (1985)
Science 229:1202-1207; by Oi et al. (1986) BioTechniques 4:214; and
by U.S. Pat. Nos. 5,585,089; 5,693,761; 5,693,762; 5,859,205; and
6,407,213. Those methods include isolating, manipulating, and
expressing the nucleic acid sequences that encode all or part of
immunoglobulin Fv variable domains from at least one of a heavy or
light chain. Such nucleic acids may be obtained from a hybridoma
producing an antibody against a predetermined target, as described
above, as well as from other sources. The recombinant DNA encoding
the humanized antibody molecule can then be cloned into an
appropriate expression vector.
Humanized antibodies may also be produced using transgenic animals
such as mice that express human heavy and light chain genes, but
are incapable of expressing the endogenous mouse immunoglobulin
heavy and light chain genes. Winter describes an exemplary CDR
grafting method that may be used to prepare the humanized
antibodies described herein (U.S. Pat. No. 5,225,539). All of the
CDRs of a particular human antibody may be replaced with at least a
portion of a non-human CDR, or only some of the CDRs may be
replaced with non-human CDRs. It is only necessary to replace the
number of CDRs required for binding of the humanized antibody to a
predetermined antigen.
A humanized antibody can be optimized by the introduction of
conservative substitutions, consensus sequence substitutions,
germline substitutions and/or back mutations. Such altered
immunoglobulin molecules can be made by any of several techniques
known in the art, (e.g., Teng et al., Proc. Natl. Acad. Sci.
U.S.A., 80: 7308-7312, 1983; Kozbor et al., Immunology Today, 4:
7279, 1983; Olsson et al., Meth. Enzymol., 92: 3-16, 1982, and EP
239 400).
The term "human antibody", "human antibody construct" and "human
binding domain" includes antibodies, antibody constructs and
binding domains having antibody regions such as variable and
constant regions or domains which correspond substantially to human
germline immunoglobulin sequences known in the art, including, for
example, those described by Kabat et al. (1991) (loc. cit.). The
human antibodies, antibody constructs or binding domains of the
invention may include amino acid residues not encoded by human
germline immunoglobulin sequences (e.g., mutations introduced by
random or site-specific mutagenesis in vitro or by somatic mutation
in vivo), for example in the CDRs, and in particular, in CDR3. The
human antibodies, antibody constructs or binding domains can have
at least one, two, three, four, five, or more positions replaced
with an amino acid residue that is not encoded by the human
germline immunoglobulin sequence. The definition of human
antibodies, antibody constructs and binding domains as used herein
also contemplates fully human antibodies, which include only
non-artificially and/or genetically altered human sequences of
antibodies as those can be derived by using technologies or systems
such as the Xenomouse.
In some embodiments, the antibody constructs of the invention are
"isolated" or "substantially pure" antibody constructs. "Isolated"
or "substantially pure", when used to describe the antibody
constructs disclosed herein, means an antibody construct that has
been identified, separated and/or recovered from a component of its
production environment. Preferably, the antibody construct is free
or substantially free of association with all other components from
its production environment. Contaminant components of its
production environment, such as that resulting from recombinant
transfected cells, are materials that would typically interfere
with diagnostic or therapeutic uses for the polypeptide, and may
include enzymes, hormones, and other proteinaceous or
non-proteinaceous solutes. The antibody constructs may e.g
constitute at least about 5%, or at least about 50% by weight of
the total protein in a given sample. It is understood that the
isolated protein may constitute from 5% to 99.9% by weight of the
total protein content, depending on the circumstances. The
polypeptide may be made at a significantly higher concentration
through the use of an inducible promoter or high expression
promoter, such that it is made at increased concentration levels.
The definition includes the production of an antibody construct in
a wide variety of organisms and/or host cells that are known in the
art. In preferred embodiments, the antibody construct will be
purified (1) to a degree sufficient to obtain at least 15 residues
of N-terminal or internal amino acid sequence by use of a spinning
cup sequenator, or (2) to homogeneity by SDS-PAGE under
non-reducing or reducing conditions using Coomassie blue or,
preferably, silver stain. Ordinarily, however, an isolated antibody
construct will be prepared by at least one purification step.
The term "binding domain" characterizes in connection with the
present invention a domain which (specifically) binds to/interacts
with/recognizes a given target epitope or a given target site on
the target molecules (antigens), here: DLL3 and CD3, respectively.
The structure and function of the first binding domain (recognizing
DLL3), and preferably also the structure and/or function of the
second binding domain (recognizing CD3), is/are based on the
structure and/or function of an antibody, e.g. of a full-length or
whole immunoglobulin molecule. According to the invention, the
first binding domain is characterized by the presence of three
light chain CDRs (i.e. CDR1, CDR2 and CDR3 of the VL region) and/or
three heavy chain CDRs (i.e. CDR1, CDR2 and CDR3 of the VH region).
The second binding domain preferably also comprises the minimum
structural requirements of an antibody which allow for the target
binding. More preferably, the second binding domain comprises at
least three light chain CDRs (i.e. CDR1, CDR2 and CDR3 of the VL
region) and/or three heavy chain CDRs (i.e. CDR1, CDR2 and CDR3 of
the VH region). It is envisaged that the first and/or second
binding domain is produced by or obtainable by phage-display or
library screening methods rather than by grafting CDR sequences
from a pre-existing (monoclonal) antibody into a scaffold.
According to the present invention, binding domains are in the form
of one or more polypeptides. Such polypeptides may include
proteinaceous parts and non-proteinaceous parts (e.g. chemical
linkers or chemical cross-linking agents such as glutaraldehyde).
Proteins (including fragments thereof, preferably biologically
active fragments, and peptides, usually having less than 30 amino
acids) comprise two or more amino acids coupled to each other via a
covalent peptide bond (resulting in a chain of amino acids). The
term "polypeptide" as used herein describes a group of molecules,
which usually consist of more than 30 amino acids. Polypeptides may
further form multimers such as dimers, trimers and higher
oligomers, i.e., consisting of more than one polypeptide molecule.
Polypeptide molecules forming such dimers, trimers etc. may be
identical or non-identical. The corresponding higher order
structures of such multimers are, consequently, termed homo- or
heterodimers, homo- or heterotrimers etc. An example for a
hereteromultimer is an antibody molecule, which, in its naturally
occurring form, consists of two identical light polypeptide chains
and two identical heavy polypeptide chains. The terms "peptide",
"polypeptide" and "protein" also refer to naturally modified
peptides/polypeptides/proteins wherein the modification is effected
e.g. by post-translational modifications like glycosylation,
acetylation, phosphorylation and the like. A "peptide",
"polypeptide" or "protein" when referred to herein may also be
chemically modified such as pegylated. Such modifications are well
known in the art and described herein below.
Preferably the binding domain which binds to DLL3 and/or the
binding domain which binds to CD3 is/are human binding domains.
Antibodies and antibody constructs comprising at least one human
binding domain avoid some of the problems associated with
antibodies or antibody constructs that possess non-human such as
rodent (e.g. murine, rat, hamster or rabbit) variable and/or
constant regions. The presence of such rodent derived proteins can
lead to the rapid clearance of the antibodies or antibody
constructs or can lead to the generation of an immune response
against the antibody or antibody construct by a patient. In order
to avoid the use of rodent derived antibodies or antibody
constructs, human or fully human antibodies/antibody constructs can
be generated through the introduction of human antibody function
into a rodent so that the rodent produces fully human
antibodies.
The ability to clone and reconstruct megabase-sized human loci in
YACs and to introduce them into the mouse germline provides a
powerful approach to elucidating the functional components of very
large or crudely mapped loci as well as generating useful models of
human disease. Furthermore, the use of such technology for
substitution of mouse loci with their human equivalents could
provide unique insights into the expression and regulation of human
gene products during development, their communication with other
systems, and their involvement in disease induction and
progression.
An important practical application of such a strategy is the
"humanization" of the mouse humoral immune system. Introduction of
human immunoglobulin (Ig) loci into mice in which the endogenous Ig
genes have been inactivated offers the opportunity to study the
mechanisms underlying programmed expression and assembly of
antibodies as well as their role in B-cell development.
Furthermore, such a strategy could provide an ideal source for
production of fully human monoclonal antibodies (mAbs)--an
important milestone towards fulfilling the promise of antibody
therapy in human disease. Fully human antibodies or antibody
constructs are expected to minimize the immunogenic and allergic
responses intrinsic to mouse or mouse-derivatized mAbs and thus to
increase the efficacy and safety of the administered
antibodies/antibody constructs. The use of fully human antibodies
or antibody constructs can be expected to provide a substantial
advantage in the treatment of chronic and recurring human diseases,
such as inflammation, autoimmunity, and cancer, which require
repeated compound administrations.
One approach towards this goal was to engineer mouse strains
deficient in mouse antibody production with large fragments of the
human Ig loci in anticipation that such mice would produce a large
repertoire of human antibodies in the absence of mouse antibodies.
Large human Ig fragments would preserve the large variable gene
diversity as well as the proper regulation of antibody production
and expression. By exploiting the mouse machinery for antibody
diversification and selection and the lack of immunological
tolerance to human proteins, the reproduced human antibody
repertoire in these mouse strains should yield high affinity
antibodies against any antigen of interest, including human
antigens. Using the hybridoma technology, antigen-specific human
mAbs with the desired specificity could be readily produced and
selected. This general strategy was demonstrated in connection with
the generation of the first XenoMouse mouse strains (see Green et
al. Nature Genetics 7:13-21 (1994)). The XenoMouse strains were
engineered with yeast artificial chromosomes (YACs) containing 245
kb and 190 kb-sized germline configuration fragments of the human
heavy chain locus and kappa light chain locus, respectively, which
contained core variable and constant region sequences. The human Ig
containing YACs proved to be compatible with the mouse system for
both rearrangement and expression of antibodies and were capable of
substituting for the inactivated mouse Ig genes. This was
demonstrated by their ability to induce B cell development, to
produce an adult-like human repertoire of fully human antibodies,
and to generate antigen-specific human mAbs. These results also
suggested that introduction of larger portions of the human Ig loci
containing greater numbers of V genes, additional regulatory
elements, and human Ig constant regions might recapitulate
substantially the full repertoire that is characteristic of the
human humoral response to infection and immunization. The work of
Green et al. was recently extended to the introduction of greater
than approximately 80% of the human antibody repertoire through
introduction of megabase sized, germline configuration YAC
fragments of the human heavy chain loci and kappa light chain loci,
respectively. See Mendez et al. Nature Genetics 15:146-156 (1997)
and U.S. patent application Ser. No. 08/759,620.
The production of the XenoMouse mice is further discussed and
delineated in U.S. patent application Ser. No. 07/466,008, Ser. No.
07/610,515, Ser. No. 07/919,297, Ser. No. 07/922,649, Ser. No.
08/031,801, Ser. No. 08/112,848, Ser. No. 08/234,145, Ser. No.
08/376,279, Ser. No. 08/430,938, Ser. No. 08/464,584, Ser. No.
08/464,582, Ser. No. 08/463,191, Ser. No. 08/462,837, Ser. No.
08/486,853, Ser. No. 08/486,857, Ser. No. 08/486,859, Ser. No.
08/462,513, Ser. No. 08/724,752, and Ser. No. 08/759,620; and U.S.
Pat. Nos. 6,162,963; 6,150,584; 6,114,598; 6,075,181, and 5,939,598
and Japanese Patent Nos. 3 068 180 B2, 3 068 506 B2, and 3 068 507
B2. See also Mendez et al. Nature Genetics 15:146-156 (1997) and
Green and Jakobovits J. Exp. Med. 188:483-495 (1998), EP 0 463 151
B1, WO 94/02602, WO 96/34096, WO 98/24893, WO 00/76310, and WO
03/47336.
In an alternative approach, others, including GenPharm
International, Inc., have utilized a "minilocus" approach. In the
minilocus approach, an exogenous Ig locus is mimicked through the
inclusion of pieces (individual genes) from the Ig locus. Thus, one
or more VH genes, one or more DH genes, one or more JH genes, a mu
constant region, and a second constant region (preferably a gamma
constant region) are formed into a construct for insertion into an
animal. This approach is described in U.S. Pat. No. 5,545,807 to
Surani et al. and U.S. Pat. Nos. 5,545,806; 5,625,825; 5,625,126;
5,633,425; 5,661,016; 5,770,429; 5,789,650; 5,814,318; 5,877,397;
5,874,299; and 6,255,458 each to Lonberg and Kay, U.S. Pat. Nos.
5,591,669 and 6,023.010 to Krimpenfort and Berns, U.S. Pat. Nos.
5,612,205; 5,721,367; and 5,789,215 to Berns et al., and U.S. Pat.
No. 5,643,763 to Choi and Dunn, and GenPharm International U.S.
patent application Ser. No. 07/574,748, Ser. No. 07/575,962, Ser.
No. 07/810,279, Ser. No. 07/853,408, Ser. No. 07/904,068, Ser. No.
07/990,860, Ser. No. 08/053,131, Ser. No. 08/096,762, Ser. No.
08/155,301, Ser. No. 08/161,739, Ser. No. 08/165,699, Ser. No.
08/209,741. See also EP 0 546 073 B1, WO 92/03918, WO 92/22645, WO
92/22647, WO 92/22670, WO 93/12227, WO 94/00569, WO 94/25585, WO
96/14436, WO 97/13852, and WO 98/24884 and U.S. Pat. No. 5,981,175.
See further Taylor et al. (1992), Chen et al. (1993), Tuaillon et
al. (1993), Choi et al. (1993), Lonberg et al. (1994), Taylor et
al. (1994), and Tuaillon et al. (1995), Fishwild et al. (1996).
Kirin has also demonstrated the generation of human antibodies from
mice in which, through microcell fusion, large pieces of
chromosomes, or entire chromosomes, have been introduced. See
European Patent Application Nos. 773 288 and 843 961. Xenerex
Biosciences is developing a technology for the potential generation
of human antibodies. In this technology, SCID mice are
reconstituted with human lymphatic cells, e.g., B and/or T cells.
Mice are then immunized with an antigen and can generate an immune
response against the antigen. See U.S. Pat. Nos. 5,476,996;
5,698,767; and 5,958,765.
Human anti-mouse antibody (HAMA) responses have led the industry to
prepare chimeric or otherwise humanized antibodies. It is however
expected that certain human anti-chimeric antibody (HACA) responses
will be observed, particularly in chronic or multi-dose
utilizations of the antibody. Thus, it would be desirable to
provide antibody constructs comprising a human binding domain
against DLL3 and/or a human binding domain against CD3 in order to
vitiate concerns and/or effects of HAMA or HACA response.
The terms "(specifically) binds to", (specifically) recognizes",
"is (specifically) directed to", and "(specifically) reacts with"
mean in accordance with this invention that a binding domain
interacts or specifically interacts with a given epitope or a given
target site on the target molecules (antigens), here: DLL3 and CD3,
respectively.
The term "epitope" refers to a site on an antigen to which a
binding domain, an antibody or immunoglobulin, or a derivative,
fragment or variant of an antibody or an immunoglobulin,
specifically binds. An "epitope" is antigenic and thus the term
epitope is sometimes also referred to herein as "antigenic
structure" or "antigenic determinant". Thus, the binding domain is
an "antigen interaction site". Said binding/interaction is also
understood to define a "specific recognition".
"Epitopes" can be formed both by contiguous amino acids or
non-contiguous amino acids juxtaposed by tertiary folding of a
protein. A "linear epitope" is an epitope where an amino acid
primary sequence comprises the recognized epitope. A linear epitope
typically includes at least 3 or at least 4, and more usually, at
least 5 or at least 6 or at least 7, for example, about 8 to about
10 amino acids in a unique sequence.
A "conformational epitope", in contrast to a linear epitope, is an
epitope wherein the primary sequence of the amino acids comprising
the epitope is not the sole defining component of the epitope
recognized (e.g., an epitope wherein the primary sequence of amino
acids is not necessarily recognized by the binding domain).
Typically a conformational epitope comprises an increased number of
amino acids relative to a linear epitope. With regard to
recognition of conformational epitopes, the binding domain
recognizes a three-dimensional structure of the antigen, preferably
a peptide or protein or fragment thereof (in the context of the
present invention, the antigenic structure for the first binding
domain is comprised within the DLL3 protein). For example, when a
protein molecule folds to form a three-dimensional structure,
certain amino acids and/or the polypeptide backbone forming the
conformational epitope become juxtaposed enabling the antibody to
recognize the epitope. Methods of determining the conformation of
epitopes include, but are not limited to, x-ray crystallography,
two-dimensional nuclear magnetic resonance (2D-NMR) spectroscopy
and site-directed spin labelling and electron paramagnetic
resonance (EPR) spectroscopy.
A method for epitope mapping is described in the following: When a
region (a contiguous amino acid stretch) in the human DLL3 protein
is exchanged/replaced with its corresponding region of a non-human
and non-primate DLL3 antigen (e.g., mouse DLL3, but others like
chicken, rat, hamster, rabbit etc. might also be conceivable), a
decrease in the binding of the binding domain is expected to occur,
unless the binding domain is cross-reactive for the non-human,
non-primate DLL3 used. Said decrease is preferably at least 10%,
20%, 30%, 40%, or 50%; more preferably at least 60%, 70%, or 80%,
and most preferably 90%, 95% or even 100% in comparison to the
binding to the respective region in the human DLL3 protein, whereby
binding to the respective region in the human DLL3 protein is set
to be 100%. It is envisaged that the aforementioned human
DLL3/non-human DLL3 chimeras are expressed in CHO cells. It is also
envisaged that the human DLL3/non-human DLL3 chimeras are fused
with a transmembrane domain and/or cytoplasmic domain of a
different membrane-bound protein such as EpCAM.
In an alternative or additional method for epitope mapping, several
truncated versions of the human DLL3 extracellular domain can be
generated in order to determine a specific region that is
recognized by a binding domain. In these truncated versions, the
different extracellular DLL3 domains/sub-domains or regions are
stepwise deleted, starting from the N-terminus. The truncated DLL3
versions that were generated and used in the context of the present
invention are depicted in FIG. 1. It is envisaged that the
truncated DLL3 versions are expressed in CHO cells. It is also
envisaged that the truncated DLL3 versions are fused with a
transmembrane domain and/or cytoplasmic domain of a different
membrane-bound protein such as EpCAM. It is also envisaged that the
truncated DLL3 versions encompass a signal peptide domain at their
N-terminus, for example a signal peptide derived from mouse IgG
heavy chain signal peptide. It is furthermore envisaged that the
truncated DLL3 versions encompass a v5 domain at their N-terminus
(following the signal peptide) which allows verifying their correct
expression on the cell surface. A decrease or a loss of binding is
expected to occur with those truncated DLL3 versions which do not
encompass any more the DLL3 region that is recognized by the
binding domain. The decrease of binding is preferably at least 10%,
20%, 30%, 40%, 50%; more preferably at least 60%, 70%, 80%, and
most preferably 90%, 95% or even 100%, whereby binding to the
entire human DLL3 protein (or its extracellular region or domain)
is set to be 100%. A method to test this loss of binding is
described in Example 2.
A further method to determine the contribution of a specific
residue of a target antigen to the recognition by a antibody
construct or binding domain is alanine scanning (see e.g. Morrison
K L & Weiss G A. Cur Opin Chem Biol. 2001 June; 5(3):302-7),
where each residue to be analyzed is replaced by alanine, e.g. via
site-directed mutagenesis. Alanine is used because of its
non-bulky, chemically inert, methyl functional group that
nevertheless mimics the secondary structure references that many of
the other amino acids possess. Sometimes bulky amino acids such as
valine or leucine can be used in cases where conservation of the
size of mutated residues is desired. Alanine scanning is a mature
technology which has been used for a long period of time.
The interaction between the binding domain and the epitope or the
region comprising the epitope implies that a binding domain
exhibits appreciable affinity for the epitope/the region comprising
the epitope on a particular protein or antigen (here: DLL3 and CD3,
respectively) and, generally, does not exhibit significant
reactivity with proteins or antigens other than DLL3 or CD3.
"Appreciable affinity" includes binding with an affinity of about
10.sup.-6 M (KD) or stronger. Preferably, binding is considered
specific when the binding affinity is about 10.sup.-12 to 10.sup.-8
M, 10.sup.-12 to 10.sup.-9 M, 10.sup.-12 to 10.sup.-19 M,
10.sup.-11 to 10.sup.-8 M, preferably of about 10.sup.-11 to
10.sup.-9 M. Whether a binding domain specifically reacts with or
binds to a target can be tested readily by, inter alia, comparing
the reaction of said binding domain with a target protein or
antigen with the reaction of said binding domain with proteins or
antigens other than DLL3 or CD3. Preferably, a binding domain of
the invention does not essentially or substantially bind to
proteins or antigens other than DLL3 or CD3 (i.e., the first
binding domain is not capable of binding to proteins other than
DLL3 and the second binding domain is not capable of binding to
proteins other than CD3).
The term "does not essentially/substantially bind" or "is not
capable of binding" means that a binding domain of the present
invention does not bind a protein or antigen other than DLL3 or
CD3, i.e., does not show reactivity of more than 30%, preferably
not more than 20%, more preferably not more than 10%, particularly
preferably not more than 9%, 8%, 7%, 6% or 5% with proteins or
antigens other than DLL3 or CD3, whereby binding to DLL3 or CD3,
respectively, is set to be 100%.
It is also envisaged that the antibody constructs of the present
invention bind to a human DLL3 isoform having one or both of the
following DLL3 point mutations: F172C and L218P. See Example 5.
Specific binding is believed to be effected by specific motifs in
the amino acid sequence of the binding domain and the antigen.
Thus, binding is achieved as a result of their primary, secondary
and/or tertiary structure as well as the result of secondary
modifications of said structures. The specific interaction of the
antigen-interaction-site with its specific antigen may result in a
simple binding of said site to the antigen. Moreover, the specific
interaction of the antigen-interaction-site with its specific
antigen may alternatively or additionally result in the initiation
of a signal, e.g. due to the induction of a change of the
conformation of the antigen, an oligomerization of the antigen,
etc.
The antibody constructs according to the invention bind to an
epitope of DLL3 which is comprised within the region as depicted in
SEQ ID NO: 260, corresponding to an amino acid stretch encompassing
the regions EGF-3 and EGF-4. Other groups of anti-DLL3 binders were
also generated and their DLL3 binding specificities were identified
during epitope mapping (see Example 2).
The largest group of generated binders recognized an epitope within
the DSL domain. However, none of those antibody constructs
fulfilled the criteria for sufficient cytotoxic activity in an
initial 18-hour .sup.51Cr-based cytotoxicity assay with stimulated
human CD8+ T cells as effector cells and hu DLL3 transfected CHO
cells as target cells.
In a further initial 48 hour FACS-based cytotoxicity assay (using
unstimulated human PBMC as effector cells and hu DLL3 transfected
CHO cells as target cells), those binders that recognized a DLL3
epitope within the N-terminus of the protein had EC50 values
between 1455 and 1580 pM. In the present context, these values are
not considered adequate for bispecific antibodies that are provided
for a therapeutic use in directing a patient's immune system, more
specifically the T cells' cytotoxic activity, against cancer
cells.
Finally, another group of binders was generated and characterized
for its cytotoxic activity in a variety of assays. The epitope
mapping of these binders revealed a specificity for a DLL3 epitope
comprised within the EGF-5 region, and to some extend also within
the EGF-6 region (for details, see Example 2). An overall view of
the data generated in the different cytotoxicity assays (see
Examples 8.3, 8.5, 8.6 and 8.7) for these binders denominated
DLL3-18, DLL3-19, DLL3-20 and DLL3-21 revealed the following: While
not all of the binders fail in all of the assays in terms of
favorable EC50 values, the entire group clearly underperforms if
compared with the antibody constructs according to the invention.
This observation is highlighted with a darker shadowing of the
results shown in Tables 6-9.
In summary, it can clearly be stated that the antibody constructs
according to the invention (which bind to an epitope of DLL3
comprised within the region as depicted in SEQ ID NO: 260) show by
far the best activity performance compared to a variety of other
groups of DLL3 binders having different epitope specificities. In
other words, the antibody constructs according to the invention
present with a favorable epitope-activity relationship, hence
supporting potent bispecific antibody construct mediated cytotoxic
activity.
In another aspect, the present invention provides a bispecific
antibody construct comprising a first binding domain which binds to
human DLL3 on the surface of a target cell and a second binding
domain which binds to human CD3 on the surface of a T cell, wherein
the first binding domain binds to an epitope of DLL3 which is
comprised within the region as depicted in SEQ ID NO: 258.
Preferably, the first binding domain of the bispecific antibody
construct of the invention comprises a VH region comprising CDR-H1,
CDR-H2 and CDR-H3 and a VL region comprising CDR-L1, CDR-L2 and
CDR-L3 selected from the group consisting of:
a) CDR-H1 as depicted in SEQ ID NO: 31, CDR-H2 as depicted in SEQ
ID NO: 32, CDR-H3 as depicted in SEQ ID NO: 33, CDR-L1 as depicted
in SEQ ID NO: 34, CDR-L2 as depicted in SEQ ID NO: 35 and CDR-L3 as
depicted in SEQ ID NO: 36;
b) CDR-H1 as depicted in SEQ ID NO: 41, CDR-H2 as depicted in SEQ
ID NO: 42, CDR-H3 as depicted in SEQ ID NO: 43, CDR-L1 as depicted
in SEQ ID NO: 44, CDR-L2 as depicted in SEQ ID NO: 45 and CDR-L3 as
depicted in SEQ ID NO: 46;
c) CDR-H1 as depicted in SEQ ID NO: 51, CDR-H2 as depicted in SEQ
ID NO: 52, CDR-H3 as depicted in SEQ ID NO: 53, CDR-L1 as depicted
in SEQ ID NO: 54, CDR-L2 as depicted in SEQ ID NO: 55 and CDR-L3 as
depicted in SEQ ID NO: 56;
d) CDR-H1 as depicted in SEQ ID NO: 61, CDR-H2 as depicted in SEQ
ID NO: 62, CDR-H3 as depicted in SEQ ID NO: 63, CDR-L1 as depicted
in SEQ ID NO: 64, CDR-L2 as depicted in SEQ ID NO: 65 and CDR-L3 as
depicted in SEQ ID NO: 66;
e) CDR-H1 as depicted in SEQ ID NO: 71, CDR-H2 as depicted in SEQ
ID NO: 72, CDR-H3 as depicted in SEQ ID NO: 73, CDR-L1 as depicted
in SEQ ID NO: 74, CDR-L2 as depicted in SEQ ID NO: 75 and CDR-L3 as
depicted in SEQ ID NO: 76;
f) CDR-H1 as depicted in SEQ ID NO: 81, CDR-H2 as depicted in SEQ
ID NO: 82, CDR-H3 as depicted in SEQ ID NO: 83, CDR-L1 as depicted
in SEQ ID NO: 84, CDR-L2 as depicted in SEQ ID NO: 85 and CDR-L3 as
depicted in SEQ ID NO: 86;
g) CDR-H1 as depicted in SEQ ID NO: 91, CDR-H2 as depicted in SEQ
ID NO: 92, CDR-H3 as depicted in SEQ ID NO: 93, CDR-L1 as depicted
in SEQ ID NO: 94, CDR-L2 as depicted in SEQ ID NO: 95 and CDR-L3 as
depicted in SEQ ID NO: 96;
h) CDR-H1 as depicted in SEQ ID NO: 101, CDR-H2 as depicted in SEQ
ID NO: 102, CDR-H3 as depicted in SEQ ID NO: 103, CDR-L1 as
depicted in SEQ ID NO: 104, CDR-L2 as depicted in SEQ ID NO: 105
and CDR-L3 as depicted in SEQ ID NO: 106; and
i) CDR-H1 as depicted in SEQ ID NO: 111, CDR-H2 as depicted in SEQ
ID NO: 112, CDR-H3 as depicted in SEQ ID NO: 113, CDR-L1 as
depicted in SEQ ID NO: 114, CDR-L2 as depicted in SEQ ID NO: 115
and CDR-L3 as depicted in SEQ ID NO: 116.
The term "variable" refers to the portions of the antibody or
immunoglobulin domains that exhibit variability in their sequence
and that are involved in determining the specificity and binding
affinity of a particular antibody (i.e., the "variable domain(s)").
The pairing of a variable heavy chain (VH) and a variable light
chain (VL) together forms a single antigen-binding site.
Variability is not evenly distributed throughout the variable
domains of antibodies; it is concentrated in sub-domains of each of
the heavy and light chain variable regions. These sub-domains are
called "hypervariable regions" or "complementarity determining
regions" (CDRs). The more conserved (i.e., non-hypervariable)
portions of the variable domains are called the "framework" regions
(FRM or FR) and provide a scaffold for the six CDRs in three
dimensional space to form an antigen-binding surface. The variable
domains of naturally occurring heavy and light chains each comprise
four FRM regions (FR1, FR2, FR3, and FR4), largely adopting a
.beta.-sheet configuration, connected by three hypervariable
regions, which form loops connecting, and in some cases forming
part of, the .beta.-sheet structure. The hypervariable regions in
each chain are held together in close proximity by the FRM and,
with the hypervariable regions from the other chain, contribute to
the formation of the antigen-binding site (see Kabat et al., loc.
cit.).
The terms "CDR", and its plural "CDRs", refer to the
complementarity determining region of which three make up the
binding character of a light chain variable region (CDR-L1, CDR-L2
and CDR-L3) and three make up the binding character of a heavy
chain variable region (CDR-H1, CDR-H2 and CDR-H3). CDRs contain
most of the residues responsible for specific interactions of the
antibody with the antigen and hence contribute to the functional
activity of an antibody molecule: they are the main determinants of
antigen specificity.
The exact definitional CDR boundaries and lengths are subject to
different classification and numbering systems. CDRs may therefore
be referred to by Kabat, Chothia, contact or any other boundary
definitions, including the numbering system described herein.
Despite differing boundaries, each of these systems has some degree
of overlap in what constitutes the so called "hypervariable
regions" within the variable sequences. CDR definitions according
to these systems may therefore differ in length and boundary areas
with respect to the adjacent framework region. See for example
Kabat (an approach based on cross-species sequence variability),
Chothia (an approach based on crystallographic studies of
antigen-antibody complexes), and/or MacCallum (Kabat et al., loc.
cit.; Chothia et al., J. Mol. Biol, 1987, 196: 901-917; and
MacCallum et al., J. Mol. Biol, 1996, 262: 732). Still another
standard for characterizing the antigen binding site is the AbM
definition used by Oxford Molecular's AbM antibody modeling
software. See, e.g., Protein Sequence and Structure Analysis of
Antibody Variable Domains. In: Antibody Engineering Lab Manual
(Ed.: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg).
To the extent that two residue identification techniques define
regions of overlapping, but not identical regions, they can be
combined to define a hybrid CDR. However, the numbering in
accordance with the so-called Kabat system is preferred.
Typically, CDRs form a loop structure that can be classified as a
canonical structure. The term "canonical structure" refers to the
main chain conformation that is adopted by the antigen binding
(CDR) loops. From comparative structural studies, it has been found
that five of the six antigen binding loops have only a limited
repertoire of available conformations. Each canonical structure can
be characterized by the torsion angles of the polypeptide backbone.
Correspondent loops between antibodies may, therefore, have very
similar three dimensional structures, despite high amino acid
sequence variability in most parts of the loops (Chothia and Lesk,
J. Mol. Biol., 1987, 196: 901; Chothia et al., Nature, 1989, 342:
877; Martin and Thornton, J. Mol. Biol, 1996, 263: 800).
Furthermore, there is a relationship between the adopted loop
structure and the amino acid sequences surrounding it. The
conformation of a particular canonical class is determined by the
length of the loop and the amino acid residues residing at key
positions within the loop, as well as within the conserved
framework (i.e., outside of the loop). Assignment to a particular
canonical class can therefore be made based on the presence of
these key amino acid residues.
The term "canonical structure" may also include considerations as
to the linear sequence of the antibody, for example, as catalogued
by Kabat (Kabat et al., loc. cit.). The Kabat numbering scheme
(system) is a widely adopted standard for numbering the amino acid
residues of an antibody variable domain in a consistent manner and
is the preferred scheme applied in the present invention as also
mentioned elsewhere herein. Additional structural considerations
can also be used to determine the canonical structure of an
antibody. For example, those differences not fully reflected by
Kabat numbering can be described by the numbering system of Chothia
et al. and/or revealed by other techniques, for example,
crystallography and two- or three-dimensional computational
modeling. Accordingly, a given antibody sequence may be placed into
a canonical class which allows for, among other things, identifying
appropriate chassis sequences (e.g., based on a desire to include a
variety of canonical structures in a library). Kabat numbering of
antibody amino acid sequences and structural considerations as
described by Chothia et al., loc. cit. and their implications for
construing canonical aspects of antibody structure, are described
in the literature. The subunit structures and three-dimensional
configurations of different classes of immunoglobulins are well
known in the art. For a review of the antibody structure, see
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,
eds. Harlow et al., 1988.
The CDR3 of the light chain and, particularly, the CDR3 of the
heavy chain may constitute the most important determinants in
antigen binding within the light and heavy chain variable regions.
In some antibody constructs, the heavy chain CDR3 appears to
constitute the major area of contact between the antigen and the
antibody. In vitro selection schemes in which CDR3 alone is varied
can be used to vary the binding properties of an antibody or
determine which residues contribute to the binding of an antigen.
Hence, CDR3 is typically the greatest source of molecular diversity
within the antibody-binding site. H3, for example, can be as short
as two amino acid residues or greater than 26 amino acids.
In a classical full-length antibody or immunoglobulin, each light
(L) chain is linked to a heavy (H) chain by one covalent disulfide
bond, while the two H chains are linked to each other by one or
more disulfide bonds depending on the H chain isotype. The CH
domain most proximal to VH is usually designated as CH1. The
constant ("C") domains are not directly involved in antigen
binding, but exhibit various effector functions, such as
antibody-dependent, cell-mediated cytotoxicity and complement
activation. The Fc region of an antibody is comprised within the
heavy chain constant domains and is for example able to interact
with cell surface located Fc receptors.
The sequence of antibody genes after assembly and somatic mutation
is highly varied, and these varied genes are estimated to encode
10.sup.10 different antibody molecules (Immunoglobulin Genes, 2nd
ed., eds. Jonio et al., Academic Press, San Diego, Calif., 1995).
Accordingly, the immune system provides a repertoire of
immunoglobulins. The term "repertoire" refers to at least one
nucleotide sequence derived wholly or partially from at least one
sequence encoding at least one immunoglobulin. The sequence(s) may
be generated by rearrangement in vivo of the V, D, and J segments
of heavy chains, and the V and J segments of light chains.
Alternatively, the sequence(s) can be generated from a cell in
response to which rearrangement occurs, e.g., in vitro stimulation.
Alternatively, part or all of the sequence(s) may be obtained by
DNA splicing, nucleotide synthesis, mutagenesis, and other methods,
see, e.g., U.S. Pat. No. 5,565,332. A repertoire may include only
one sequence or may include a plurality of sequences, including
ones in a genetically diverse collection.
A preferred antibody construct according to the invention can also
be defined as a bispecific antibody construct comprising a first
(preferably human) binding domain which binds to human DLL3 on the
surface of a target cell and a second binding domain which binds to
human CD3 on the surface of a T cell, wherein the first binding
domain binds to the same epitope of DLL3 as an antibody selected
from the group consisting of DLL3-4, DLL3-5, DLL3-6, DLL3-7,
DLL3-8, DLL3-9, and DLL3-10, i.e., an antibody comprising a VH
region comprising CDR-H1, CDR-H2 and CDR-H3 and a VL region
comprising CDR-L1, CDR-L2 and CDR-L3 selected from the group
consisting of:
a) CDR-H1 as depicted in SEQ ID NO: 31, CDR-H2 as depicted in SEQ
ID NO: 32, CDR-H3 as depicted in SEQ ID NO: 33, CDR-L1 as depicted
in SEQ ID NO: 34, CDR-L2 as depicted in SEQ ID NO: 35 and CDR-L3 as
depicted in SEQ ID NO: 36;
b) CDR-H1 as depicted in SEQ ID NO: 41, CDR-H2 as depicted in SEQ
ID NO: 42, CDR-H3 as depicted in SEQ ID NO: 43, CDR-L1 as depicted
in SEQ ID NO: 44, CDR-L2 as depicted in SEQ ID NO: 45 and CDR-L3 as
depicted in SEQ ID NO: 46;
c) CDR-H1 as depicted in SEQ ID NO: 51, CDR-H2 as depicted in SEQ
ID NO: 52, CDR-H3 as depicted in SEQ ID NO: 53, CDR-L1 as depicted
in SEQ ID NO: 54, CDR-L2 as depicted in SEQ ID NO: 55 and CDR-L3 as
depicted in SEQ ID NO: 56;
d) CDR-H1 as depicted in SEQ ID NO: 61, CDR-H2 as depicted in SEQ
ID NO: 62, CDR-H3 as depicted in SEQ ID NO: 63, CDR-L1 as depicted
in SEQ ID NO: 64, CDR-L2 as depicted in SEQ ID NO: 65 and CDR-L3 as
depicted in SEQ ID NO: 66;
e) CDR-H1 as depicted in SEQ ID NO: 71, CDR-H2 as depicted in SEQ
ID NO: 72, CDR-H3 as depicted in SEQ ID NO: 73, CDR-L1 as depicted
in SEQ ID NO: 74, CDR-L2 as depicted in SEQ ID NO: 75 and CDR-L3 as
depicted in SEQ ID NO: 76;
f) CDR-H1 as depicted in SEQ ID NO: 81, CDR-H2 as depicted in SEQ
ID NO: 82, CDR-H3 as depicted in SEQ ID NO: 83, CDR-L1 as depicted
in SEQ ID NO: 84, CDR-L2 as depicted in SEQ ID NO: 85 and CDR-L3 as
depicted in SEQ ID NO: 86; and
g) CDR-H1 as depicted in SEQ ID NO: 91, CDR-H2 as depicted in SEQ
ID NO: 92, CDR-H3 as depicted in SEQ ID NO: 93, CDR-L1 as depicted
in SEQ ID NO: 94, CDR-L2 as depicted in SEQ ID NO: 95 and CDR-L3 as
depicted in SEQ ID NO: 96.
Whether or not an antibody construct binds to the same epitope of
DLL3 as another given antibody construct can be measured e.g. by
epitope mapping with chimeric or truncated target molecules, e.g.
as described herein above and in Example 2.
A preferred antibody construct according to the invention can also
be defined as a bispecific antibody construct comprising a first
(preferably human) binding domain which binds to human DLL3 on the
surface of a target cell and a second binding domain which binds to
human CD3 on the surface of a T cell, wherein the first binding
domain competes for binding with an antibody selected from the
group consisting of DLL3-4, DLL3-5, DLL3-6, DLL3-7, DLL3-8, DLL3-9,
and DLL3-10, i.e., an antibody comprising a VH region comprising
CDR-H1, CDR-H2 and CDR-H3 and a VL region comprising CDR-L1, CDR-L2
and CDR-L3 selected from the group consisting of those described
above.
Whether or not an antibody construct competes for binding with
another given antibody construct can be measured in a competition
assay such as a competitive ELISA or a cell-based competition
assay. Avidin-coupled microparticles (beads) can also be used.
Similar to an avidin-coated ELISA plate, when reacted with a
biotinylated protein, each of these beads can be used as a
substrate on which an assay can be performed. Antigen is coated
onto a bead and then precoated with the first antibody. The second
antibody is added and any additional binding is determined.
Read-out occurs via flow cytometry.
In one embodiment of the invention, the first binding domain of the
antibody construct of the invention comprises a VH region selected
from the group consisting of those depicted in SEQ ID NO: 37, SEQ
ID NO: 47, SEQ ID NO: 57, SEQ ID NO: 67, SEQ ID NO: 77, SEQ ID NO:
87, SEQ ID NO: 97, SEQ ID NO: 107, SEQ ID NO: 117, SEQ ID NO: 435
and SEQ ID NO: 529.
In a further embodiment, the first binding domain of the antibody
construct of the invention comprises a VL region selected from the
group consisting of those depicted in SEQ ID NO: 38, SEQ ID NO: 48,
SEQ ID NO: 58, SEQ ID NO: 68, SEQ ID NO: 78, SEQ ID NO: 88, SEQ ID
NO: 98, SEQ ID NO: 108, SEQ ID NO: 118, SEQ ID NO: 436 and SEQ ID
NO: 530.
In another embodiment, the first binding domain of the antibody
construct of the invention comprises a VH region and a VL region
selected from the group consisting of pairs of a VH region and a VL
region as depicted in SEQ ID NOs: 37+38; SEQ ID NOs: 47+48; SEQ ID
NOs: 57+58; SEQ ID NOs: 67+68; SEQ ID NOs: 77+78; SEQ ID NOs:
87+88; SEQ ID NOs: 97+98; SEQ ID NOs: 107+108; SEQ ID NOs: 117+118;
SEQ ID NOs: 435+436; and SEQ ID Nos: 529+530.
In yet a further embodiment, the first binding domain of the
antibody construct of the invention comprises a polypeptide
selected from the group consisting of those depicted in SEQ ID NO:
39, SEQ ID NO: 49, SEQ ID NO: 59, SEQ ID NO: 69, SEQ ID NO: 79, SEQ
ID NO: 89, SEQ ID NO: 99, SEQ ID NO: 109, SEQ ID NO: 119, SEQ ID
NO: 437 and SEQ ID NO: 531.
The above first binding domains (which are specified by their CDRs,
VH region and VL region and combinations thereof) characterize as
binding domains which bind to a DLL3 epitope comprised within the
region as depicted in SEQ ID NO: 258.
The term "bispecific" as used herein refers to an antibody
construct which is "at least bispecific", i.e., it comprises at
least a first binding domain and a second binding domain, wherein
the first binding domain binds to one antigen or target (here:
DLL3), and the second binding domain binds to another antigen or
target (here: CD3). Accordingly, antibody constructs according to
the invention comprise specificities for at least two different
antigens or targets. The term "bispecific antibody construct" of
the invention also encompasses multispecific antibody constructs
such as trispecific antibody constructs, the latter ones including
three binding domains, or constructs having more than three (e.g.
four, five . . . ) specificites.
Given that the antibody constructs according to the invention are
(at least) bispecific, they do not occur naturally and they are
markedly different from naturally occurring products. A
"bispecific" antibody construct or immunoglobulin is hence an
artificial hybrid antibody or immunoglobulin having at least two
distinct binding sites with different specificities. Bispecific
antibody constructs can be produced by a variety of methods
including fusion of hybridomas or linking of Fab' fragments. See,
e.g., Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321
(1990).
The at least two binding domains and the variable domains of the
antibody construct of the present invention may or may not comprise
peptide linkers (spacer peptides). The term "peptide linker"
comprises in accordance with the present invention an amino acid
sequence by which the amino acid sequences of one (variable and/or
binding) domain and another (variable and/or binding) domain of the
antibody construct of the invention are linked with each other. An
essential technical feature of such peptide linker is that it does
not comprise any polymerization activity. Among the suitable
peptide linkers are those described in U.S. Pat. Nos. 4,751,180 and
4,935,233 or WO 88/09344. The peptide linkers can also be used to
attach other domains or modules or regions (such as half-life
extending domains) to the antibody construct of the invention.
In the event that a linker is used, this linker is preferably of a
length and sequence sufficient to ensure that each of the first and
second domains can, independently from one another, retain their
differential binding specificities. For peptide linkers which
connect the at least two binding domains (or two variable domains)
in the antibody construct of the invention, those peptide linkers
are preferred which comprise only a few number of amino acid
residues, e.g. 12 amino acid residues or less. Thus, peptide
linkers of 12, 11, 10, 9, 8, 7, 6 or 5 amino acid residues are
preferred. An envisaged peptide linker with less than 5 amino acids
comprises 4, 3, 2 or one amino acid(s), wherein Gly-rich linkers
are preferred. A particularly preferred "single" amino acid in the
context of said "peptide linker" is Gly. Accordingly, said peptide
linker may consist of the single amino acid Gly. Another preferred
embodiment of a peptide linker is characterized by the amino acid
sequence Gly-Gly-Gly-Gly-Ser, i.e. Gly4Ser (SEQ ID NO: 286), or
polymers thereof, i.e. (Gly4Ser)x, where x is an integer of 1 or
greater (e.g. 2 or 3). Preferred linkers are depicted in SEQ ID
NOs: 285-293. The characteristics of said peptide linker, which
comprise the absence of the promotion of secondary structures, are
known in the art and are described e.g. in Dall'Acqua et al.
(Biochem. (1998) 37, 9266-9273), Cheadle et al. (Mol Immunol (1992)
29, 21-30) and Raag and Whitlow (FASEB (1995) 9(1), 73-80). Peptide
linkers which furthermore do not promote any secondary structures
are preferred. The linkage of said domains to each other can be
provided, e.g., by genetic engineering, as described in the
examples. Methods for preparing fused and operatively linked
bispecific single chain constructs and expressing them in mammalian
cells or bacteria are well-known in the art (e.g. WO 99/54440 or
Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
2001).
As described herein above, the invention provides a preferred
embodiment wherein the antibody construct is in a format selected
from the group consisting of (scFv)2, scFv-single domain mAb,
diabodies and oligomers of any of the afermentioned formats. The
term "is in a format" does not exclude that the construct can be
further modified, e.g. by attachment or fusion to other moieties,
as described herein.
According to a particularly preferred embodiment, and as documented
in the appended examples, the antibody construct of the invention
is a "bispecific single chain antibody construct", more prefereably
a bispecific "single chain Fv" (scFv). Although the two domains of
the Fv fragment, VL and VH, are coded for by separate genes, they
can be joined, using recombinant methods, by a synthetic linker--as
described hereinbefore--that enables them to be made as a single
protein chain in which the VL and VH regions pair to form a
monovalent molecule; see e.g., Huston et al. (1988) Proc. Natl.
Acad. Sci USA 85:5879-5883). These antibody fragments are obtained
using conventional techniques known to those with skill in the art,
and the fragments are evaluated for function in the same manner as
are whole or full-length antibodies. A single-chain variable
fragment (scFv) is hence a fusion protein of the variable region of
the heavy chain (VH) and of the light chain (VL) of
immunoglobulins, usually connected with a short linker peptide of
about ten to about 25 amino acids, preferably about 15 to 20 amino
acids. The linker is usually rich in glycine for flexibility, as
well as serine or threonine for solubility, and can either connect
the N-terminus of the VH with the C-terminus of the VL, or vice
versa. This protein retains the specificity of the original
immunoglobulin, despite removal of the constant regions and
introduction of the linker.
Bispecific single chain molecules are known in the art and are
described in WO 99/54440, Mack, J. Immunol. (1997), 158, 3965-3970,
Mack, PNAS, (1995), 92, 7021-7025, Kufer, Cancer Immunol.
Immunother., (1997), 45, 193-197, Loffler, Blood, (2000), 95, 6,
2098-2103, Bruhl, Immunol., (2001), 166, 2420-2426, Kipriyanov, J.
Mol. Biol., (1999), 293, 41-56. Techniques described for the
production of single chain antibodies (see, inter alia, U.S. Pat.
No. 4,946,778, Kontermann and Dubel (2010), loc. cit. and Little
(2009), loc. cit.) can be adapted to produce single chain antibody
constructs specifically recognizing (an) elected target(s).
Bivalent (also called divalent) or bispecific single-chain variable
fragments (bi-scFvs or di-scFvs having the format (scFv)2 can be
engineered by linking two scFv molecules (e.g. with linkers as
described hereinbefore). If these two scFv molecules have the same
binding specificity, the resulting (scFv)2 molecule will preferably
be called bivalent (i.e. it has two valences for the same target
epitope). If the two scFv molecules have different binding
specificities, the resulting (scFv)2 molecule will preferably be
called bispecific. The linking can be done by producing a single
peptide chain with two VH regions and two VL regions, yielding
tandem scFvs (see e.g. Kufer P. et al., (2004) Trends in
Biotechnology 22(5):238-244). Another possibility is the creation
of scFv molecules with linker peptides that are too short for the
two variable regions to fold together (e.g. about five amino
acids), forcing the scFvs to dimerize. This type is known as
diabodies (see e.g. Hollinger, Philipp et al., (July 1993)
Proceedings of the National Academy of Sciences of the United
States of America 90 (14): 6444-8.).
According to a further preferred embodiment of the antibody
construct of the invention the heavy chain (VH) and the light chain
(VL) of a binding domain (binding either to the target antigen DLL3
or to CD3) are not directly connected via a peptide linker as
described above, but the binding domains are formed as described
for the diabody. Thus, the VH of the CD3 binding domain may be
fused to the VL of the DLL3 binding domain via a peptide linker,
and the VH of the DLL3 binding domain is fused to the VL of the CD3
binding domain via such peptide linker.
Single domain antibodies comprise merely one (monomeric) antibody
variable domain which is able to bind selectively to a specific
antigen, independently of other V regions or domains. The first
single domain antibodies were engineered from havy chain antibodies
found in camelids, and these are called VHH fragments.
Cartilaginous fishes also have heavy chain antibodies (IgNAR) from
which single domain antibodies called VNAR fragments can be
obtained. An alternative approach is to split the dimeric variable
domains from common immunoglobulins e.g. from humans or rodents
into monomers, hence obtaining VH or VL as a single domain Ab.
Although most research into single domain antibodies is currently
based on heavy chain variable domains, nanobodies derived from
light chains have also been shown to bind specifically to target
epitopes. Examples of single domain antibodies are called sdAb,
nanobodies or single variable domain antibodies.
A (single domain mAb)2 is hence a monoclonal antibody construct
composed of (at least) two single domain monoclonal antibodies,
which are individually selected from the group comprising VH, VL,
VHH and VNAR. The linker is preferably in the form of a peptide
linker. Similarly, an "scFv-single domain mAb" is a monoclonal
antibody construct composed of at least one single domain antibody
as described above and one scFv molecule as described above. Again,
the linker is preferably in the form of a peptide linker.
It is furthermore envisaged that the present invention provides a
bispecific antibody construct comprising a first binding domain
which binds to human DLL3 on the surface of a target cell and a
second binding domain which binds to human CD3 on the surface of a
T cell, wherein the first binding domain binds to an epitope of
DLL3 which is comprised within the region as depicted in SEQ ID NO:
259.
Accordingly, in a further aspect of the invention, the first
binding domain of the bispecific antibody construct comprises a VH
region comprising CDR-H1, CDR-H2 and CDR-H3 and a VL region
comprising CDR-L1, CDR-L2 and CDR-L3 selected from the group
consisting of:
a) CDR-H1 as depicted in SEQ ID NO: 121, CDR-H2 as depicted in SEQ
ID NO: 122, CDR-H3 as depicted in SEQ ID NO: 123, CDR-L1 as
depicted in SEQ ID NO: 124, CDR-L2 as depicted in SEQ ID NO: 125
and CDR-L3 as depicted in SEQ ID NO: 126;
b) CDR-H1 as depicted in SEQ ID NO: 131, CDR-H2 as depicted in SEQ
ID NO: 132, CDR-H3 as depicted in SEQ ID NO: 133, CDR-L1 as
depicted in SEQ ID NO: 134, CDR-L2 as depicted in SEQ ID NO: 135
and CDR-L3 as depicted in SEQ ID NO: 136;
c) CDR-H1 as depicted in SEQ ID NO: 141, CDR-H2 as depicted in SEQ
ID NO: 142, CDR-H3 as depicted in SEQ ID NO: 143, CDR-L1 as
depicted in SEQ ID NO: 144, CDR-L2 as depicted in SEQ ID NO: 145
and CDR-L3 as depicted in SEQ ID NO: 146;
d) CDR-H1 as depicted in SEQ ID NO: 151, CDR-H2 as depicted in SEQ
ID NO: 152, CDR-H3 as depicted in SEQ ID NO: 153, CDR-L1 as
depicted in SEQ ID NO: 154, CDR-L2 as depicted in SEQ ID NO: 155
and CDR-L3 as depicted in SEQ ID NO: 156; and e) CDR-H1 as depicted
in SEQ ID NO: 161, CDR-H2 as depicted in SEQ ID NO: 162, CDR-H3 as
depicted in SEQ ID NO: 163, CDR-L1 as depicted in SEQ ID NO: 164,
CDR-L2 as depicted in SEQ ID NO: 165 and CDR-L3 as depicted in SEQ
ID NO: 166;
f) CDR-H1 as depicted in SEQ ID NO: 131, CDR-H2 as depicted in SEQ
ID NO: 439, CDR-H3 as depicted in SEQ ID NO: 133, CDR-L1 as
depicted in SEQ ID NO: 134, CDR-L2 as depicted in SEQ ID NO: 135
and CDR-L3 as depicted in SEQ ID NO: 136;
g) CDR-H1 as depicted in SEQ ID NO: 131, CDR-H2 as depicted in SEQ
ID NO: 440, CDR-H3 as depicted in SEQ ID NO: 133, CDR-L1 as
depicted in SEQ ID NO: 134, CDR-L2 as depicted in SEQ ID NO: 135
and CDR-L3 as depicted in SEQ ID NO: 136;
h) CDR-H1 as depicted in SEQ ID NO: 131, CDR-H2 as depicted in SEQ
ID NO: 132, CDR-H3 as depicted in SEQ ID NO: 133, CDR-L1 as
depicted in SEQ ID NO: 441, CDR-L2 as depicted in SEQ ID NO: 135
and CDR-L3 as depicted in SEQ ID NO: 136;
i) CDR-H1 as depicted in SEQ ID NO: 131, CDR-H2 as depicted in SEQ
ID NO: 132, CDR-H3 as depicted in SEQ ID NO: 133, CDR-L1 as
depicted in SEQ ID NO: 442, CDR-L2 as depicted in SEQ ID NO: 135
and CDR-L3 as depicted in SEQ ID NO: 136;
j) CDR-H1 as depicted in SEQ ID NO: 131, CDR-H2 as depicted in SEQ
ID NO: 132, CDR-H3 as depicted in SEQ ID NO: 133, CDR-L1 as
depicted in SEQ ID NO: 443, CDR-L2 as depicted in SEQ ID NO: 135
and CDR-L3 as depicted in SEQ ID NO: 136;
k) CDR-H1 as depicted in SEQ ID NO: 131, CDR-H2 as depicted in SEQ
ID NO: 132, CDR-H3 as depicted in SEQ ID NO: 133, CDR-L1 as
depicted in SEQ ID NO: 444, CDR-L2 as depicted in SEQ ID NO: 135
and CDR-L3 as depicted in SEQ ID NO: 136;
l) CDR-H1 as depicted in SEQ ID NO: 131, CDR-H2 as depicted in SEQ
ID NO: 439, CDR-H3 as depicted in SEQ ID NO: 133, CDR-L1 as
depicted in SEQ ID NO: 441, CDR-L2 as depicted in SEQ ID NO: 135
and CDR-L3 as depicted in SEQ ID NO: 136; and m) CDR-H1 as depicted
in SEQ ID NO: 131, CDR-H2 as depicted in SEQ ID NO: 440, CDR-H3 as
depicted in SEQ ID NO: 133, CDR-L1 as depicted in SEQ ID NO: 442,
CDR-L2 as depicted in SEQ ID NO: 135 and CDR-L3 as depicted in SEQ
ID NO: 136.
In one embodiment, the first binding domain of the antibody
construct of the invention comprises a VH region selected from the
group consisting of those depicted in SEQ ID NO: 127, SEQ ID NO:
137, SEQ ID NO: 147, SEQ ID NO: 157, SEQ ID NO: 167, SEQ ID NO:
445, SEQ ID NO: 446, SEQ ID NO: 447, SEQ ID NO: 448, SEQ ID NO:
449, SEQ ID NO: 450, SEQ ID NO: 451, SEQ ID NO: 452, SEQ ID NO:
453, SEQ ID NO: 454, and SEQ ID NO: 455.
In a further embodiment, the first binding domain of the antibody
construct of the invention comprises a VL region selected from the
group consisting of those depicted in SEQ ID NO: 128, SEQ ID NO:
138, SEQ ID NO: 148, SEQ ID NO: 158, SEQ ID NO: 168, SEQ ID NO:
456, SEQ ID NO: 457, SEQ ID NO: 458, SEQ ID NO: 459, SEQ ID NO:
460, SEQ ID NO: 461, SEQ ID NO: 462, SEQ ID NO: 463, SEQ ID NO:
464, SEQ ID NO: 465, SEQ ID NO: 466, SEQ ID NO: 467, SEQ ID NO:
468, SEQ ID NO: 469, and SEQ ID NO: 470.
In another embodiment, the first binding domain of the antibody
construct of the invention comprises a VH region and a VL region
selected from the group consisting of pairs of a VH region and a VL
region as depicted in SEQ ID NOs: 127+128; SEQ ID NOs: 137+138; SEQ
ID NOs: 147+148; SEQ ID NOs: 157+158; SEQ ID NOs: 167+168; SEQ ID
NOs 137+456; SEQ ID NOs 137+457; SEQ ID NOs 137+458; SEQ ID NOs
137+459; SEQ ID NOs 137+460; SEQ ID NOs 445+138; SEQ ID NOs
446+138; SEQ ID NOs 447+138; SEQ ID NOs 445+460; SEQ ID NOs
448+461; SEQ ID NOs 449+462; SEQ ID NOs 450+463; SEQ ID NOs
450+464; SEQ ID NOs 450+465; SEQ ID NOs 450+466; SEQ ID NOs
450+467; SEQ ID NOs 450+468; SEQ ID NOs 451+463; SEQ ID NOs
452+463; SEQ ID NOs 453+463; SEQ ID NOs 451+468; SEQ ID NOs
454+469; and SEQ ID NOs 455+470.
In a further embodiment, the first binding domain of the antibody
construct of the invention comprises a polypeptide selected from
the group consisting of those depicted in SEQ ID NO: 129, SEQ ID
NO: 139, SEQ ID NO: 149, SEQ ID NO: 159, SEQ ID NO: 169, SEQ ID NO:
471, SEQ ID NO: 472, SEQ ID NO: 473, SEQ ID NO: 474, SEQ ID NO:
475, SEQ ID NO: 476, SEQ ID NO: 477, SEQ ID NO: 478, SEQ ID NO:
479, SEQ ID NO: 480, SEQ ID NO: 481, SEQ ID NO: 482, SEQ ID NO:
483, SEQ ID NO: 484, SEQ ID NO: 485, SEQ ID NO: 486, SEQ ID NO:
487, SEQ ID NO: 488, SEQ ID NO: 489, SEQ ID NO: 490, SEQ ID NO:
491, SEQ ID NO: 492, and SEQ ID NO: 493.
The above first binding domains (which are specified by their CDRs,
VH region and VL region and combinations thereof) characterize as
binding domains which bind to a DLL3 epitope comprised within the
region as depicted in SEQ ID NO: 259.
Another preferred antibody construct according to the invention can
also be defined as a bispecific antibody construct comprising a
first (preferably human) binding domain which binds to human DLL3
on the surface of a target cell and a second binding domain which
binds to human CD3 on the surface of a T cell, wherein the first
binding domain binds to the same epitope of DLL3 as an antibody
selected from the group consisting of DLL3-13, DLL3-14, and
DLL3-15, i.e., an antibody comprising a VH region comprising
CDR-H1, CDR-H2 and CDR-H3 and a VL region comprising CDR-L1, CDR-L2
and CDR-L3 selected from the group consisting of:
a) CDR-H1 as depicted in SEQ ID NO: 121, CDR-H2 as depicted in SEQ
ID NO: 122, CDR-H3 as depicted in SEQ ID NO: 123, CDR-L1 as
depicted in SEQ ID NO: 124, CDR-L2 as depicted in SEQ ID NO: 125
and CDR-L3 as depicted in SEQ ID NO: 126;
b) CDR-H1 as depicted in SEQ ID NO: 131, CDR-H2 as depicted in SEQ
ID NO: 132, CDR-H3 as depicted in SEQ ID NO: 133, CDR-L1 as
depicted in SEQ ID NO: 134, CDR-L2 as depicted in SEQ ID NO: 135
and CDR-L3 as depicted in SEQ ID NO: 136; and
c) CDR-H1 as depicted in SEQ ID NO: 141, CDR-H2 as depicted in SEQ
ID NO: 142, CDR-H3 as depicted in SEQ ID NO: 143, CDR-L1 as
depicted in SEQ ID NO: 144, CDR-L2 as depicted in SEQ ID NO: 145
and CDR-L3 as depicted in SEQ ID NO: 146.
Another preferred antibody construct according to the invention can
also be defined as a bispecific antibody construct comprising a
first (preferably human) binding domain which binds to human DLL3
on the surface of a target cell and a second binding domain which
binds to human CD3 on the surface of a T cell, wherein the first
binding domain competes for binding with an antibody selected from
the group consisting of DLL3-13, DLL3-14, and DLL3-15, i.e., an
antibody comprising a VH region comprising CDR-H1, CDR-H2 and
CDR-H3 and a VL region comprising CDR-L1, CDR-L2 and CDR-L3
selected from the group consisting of those described above.
It is also envisaged that the antibody construct of the invention
has, in addition to its function to bind to the target molecules
DLL3 and CD3, a further function. In this format, the antibody
construct is a trifunctional or multifunctional antibody construct
by targeting target cells through binding to DLL3, mediating
cytotoxic T cell activity through CD3 binding and providing a
further function such as a fully functional Fc constant domain
mediating antibody-dependent cellular cytotoxicity through
recruitment of effector cells like NK cells, a label (fluorescent
etc.), a therapeutic agent such as a toxin or radionuclide, and/or
means to enhance serum half-life, etc.
Examples for means to extend serum half-life of the antibody
constructs of the invention include peptides, proteins or domains
of proteins, which are fused or otherwise attached to the antibody
constructs. The group of peptides, proteins or protein domains
includes peptides binding to other proteins with preferred
pharmacokinetic profile in the human body such as serum albumin
(see WO 2009/127691). An alternative concept of such half-life
extending peptides includes peptides binding to the neonatal Fc
receptor (FcRn, see WO 2007/098420), which can also be used in the
constructs of the present invention. The concept of attaching
larger domains of proteins or complete proteins includes e.g. the
fusion of human serum albumin, variants or mutants of human serum
albumin (see WO 2011/051489, WO 2012/059486, WO 2012/150319, WO
2013/135896, WO 2014/072481, WO 2013/075066) or domains thereof as
well as the fusion of constant region of immunoglobulins (Fc
domains) and variants thereof. Such variants of Fc domains may be
optimized/modified in order to allow the desired pairing of dimers
or mulimers, to abolish Fc receptor binding (e.g. the Fc.gamma.
receptor) or for other reasons. A further concept known in the art
to extend the half-life of small protein compounds in the human
body is the pegylation of those compounds such as the antibody
construct of the present invention.
In a preferred embodiment, the bispecific antibody constructs
according to the invention may be linked (e.g. via peptide bond)
with a fusion partner (such as a protein or polypeptide or
peptide), e.g. for the purpose of extending the construct's serum
half-life. These fusion partners can be selected from human serum
albumin ("HSA" or "HALB") as wells as sequence variants thereof,
peptides binding to HSA, peptides binding to FcRn ("FcRn BP"), or
constructs comprising an (antibody derived) Fc region. Exemplary
sequences of these fusion partners are depticed in SEQ ID NOs:
295-341. In general, the fusion partners may be linked to the
N-terminus or to the C-terminus of the bispecific antibody
constructs according to the invention, either directly (e.g. via
peptide bond) or through a peptide linker such as (GGGGS)n (wherein
"n" is an integer of 2 or greater, e.g. 2 or 3 or 4). Suitable
peptide linkers are depticed in SEQ ID NOs: 285-293.
Hence, a preferred antibody construct according to the present
invention comprises:
(a) a polypeptide comprising in the following order starting from
the N-terminus:
a polypeptide having an amino acid sequence selected from the group
consisting of SEQ ID NO: 39, SEQ ID NO: 49, SEQ ID NO: 59, SEQ ID
NO: 69, SEQ ID NO: 79, SEQ ID NO: 89, SEQ ID NO: 99, SEQ ID NO:
109, SEQ ID NO: 119, SEQ ID NO: 129, SEQ ID NO: 139, SEQ ID NO:
149, SEQ ID NO: 159, SEQ ID NO: 169, SEQ ID NO: 437, SEQ ID NO:
471, SEQ ID NO: 472, SEQ ID NO: 473, SEQ ID NO: 474, SEQ ID NO:
475, SEQ ID NO: 476, SEQ ID NO: 477, SEQ ID NO: 478, SEQ ID NO:
479, SEQ ID NO: 480, SEQ ID NO: 481, SEQ ID NO: 482, SEQ ID NO:
483, SEQ ID NO: 484, SEQ ID NO: 485, SEQ ID NO: 486, SEQ ID NO:
487, SEQ ID NO: 488, SEQ ID NO: 489, SEQ ID NO: 490, SEQ ID NO:
491, SEQ ID NO: 492, SEQ ID NO: 493, and SEQ ID NO: 531;
a peptide linker having an amino acid sequence selected from the
group consisting of SEQ ID NOs: 285-293; and
a polypeptide having an amino acid sequence selected from the group
consisting of SEQ ID NO: 350, SEQ ID NO: 359, SEQ ID NO: 368, SEQ
ID NO: 377, SEQ ID NO: 386, SEQ ID NO: 395, SEQ ID NO: 404, SEQ ID
NO: 413, SEQ ID NO: 422, SEQ ID NO: 431, and SEQ ID NO: 434;
and
optionally a His-tag, such as the one depicted in SEQ ID NO:
294;
(b) a polypeptide comprising in the following order starting from
the N-terminus:
a polypeptide having an amino acid sequence selected from the group
consisting of SEQ ID NO: 39, SEQ ID NO: 49, SEQ ID NO: 59, SEQ ID
NO: 69, SEQ ID NO: 79, SEQ ID NO: 89, SEQ ID NO: 99, SEQ ID NO:
109, SEQ ID NO: 119, SEQ ID NO: 129, SEQ ID NO: 139, SEQ ID NO:
149, SEQ ID NO: 159, SEQ ID NO: 169, SEQ ID NO: 437, SEQ ID NO:
471, SEQ ID NO: 472, SEQ ID NO: 473, SEQ ID NO: 474, SEQ ID NO:
475, SEQ ID NO: 476, SEQ ID NO: 477, SEQ ID NO: 478, SEQ ID NO:
479, SEQ ID NO: 480, SEQ ID NO: 481, SEQ ID NO: 482, SEQ ID NO:
483, SEQ ID NO: 484, SEQ ID NO: 485, SEQ ID NO: 486, SEQ ID NO:
487, SEQ ID NO: 488, SEQ ID NO: 489, SEQ ID NO: 490, SEQ ID NO:
491, SEQ ID NO: 492, SEQ ID NO: 493, and SEQ ID NO: 531;
a peptide linker having an amino acid sequence selected from the
group consisting of SEQ ID NOs: 285-293;
a polypeptide having an amino acid sequence selected from the group
consisting of SEQ ID NO: 350, SEQ ID NO: 359, SEQ ID NO: 368, SEQ
ID NO: 377, SEQ ID NO: 386, SEQ ID NO: 395, SEQ ID NO: 404, SEQ ID
NO: 413, SEQ ID NO: 422, SEQ ID NO: 431, and SEQ ID NO: 434;
optionally a peptide linker having an amino acid sequence selected
from the group consisting of SEQ ID NOs: 285-293;
a polypeptide having an amino acid sequence selected from the group
consisting of SEQ ID NOs: 295 and 301-330; and
optionally a His-tag, such as the one depicted in SEQ ID NO:
294;
(c) a polypeptide comprising in the following order starting from
the N-terminus:
a polypeptide having the amino acid sequence QRFVTGHFGGLX1PANG (SEQ
ID NO: 296) wherein X1 is Y or H; and
a polypeptide having an amino acid sequence selected from the group
consisting of SEQ ID NO: 39, SEQ ID NO: 49, SEQ ID NO: 59, SEQ ID
NO: 69, SEQ ID NO: 79, SEQ ID NO: 89, SEQ ID NO: 99, SEQ ID NO:
109, SEQ ID NO: 119, SEQ ID NO: 129, SEQ ID NO: 139, SEQ ID NO:
149, SEQ ID NO: 159, SEQ ID NO: 169, SEQ ID NO: 437, SEQ ID NO:
471, SEQ ID NO: 472, SEQ ID NO: 473, SEQ ID NO: 474, SEQ ID NO:
475, SEQ ID NO: 476, SEQ ID NO: 477, SEQ ID NO: 478, SEQ ID NO:
479, SEQ ID NO: 480, SEQ ID NO: 481, SEQ ID NO: 482, SEQ ID NO:
483, SEQ ID NO: 484, SEQ ID NO: 485, SEQ ID NO: 486, SEQ ID NO:
487, SEQ ID NO: 488, SEQ ID NO: 489, SEQ ID NO: 490, SEQ ID NO:
491, SEQ ID NO: 492, SEQ ID NO: 493, and SEQ ID NO: 531;
a peptide linker having an amino acid sequence selected from the
group consisting of SEQ ID NOs: 285-293;
a polypeptide having an amino acid sequence selected from the group
consisting of SEQ ID NO: 350, SEQ ID NO: 359, SEQ ID NO: 368, SEQ
ID NO: 377, SEQ ID NO: 386, SEQ ID NO: 395, SEQ ID NO: 404, SEQ ID
NO: 413, SEQ ID NO: 422, SEQ ID NO: 431, and SEQ ID NO: 434;
a polypeptide having the amino acid sequence QRFVTGHFGGLHPANG (SEQ
ID NO: 298) or QRFCTGHFGGLHPCNG (SEQ ID NO: 300); and
optionally a His-tag, such as the one depicted in SEQ ID NO:
294;
(d) a polypeptide comprising in the following order starting from
the N-terminus
a polypeptide having an amino acid sequence selected from the group
consisting of SEQ ID NO: 348, SEQ ID NO: 357, SEQ ID NO: 366, SEQ
ID NO: 375, SEQ ID NO: 384, SEQ ID NO: 393, SEQ ID NO: 402, SEQ ID
NO: 411, SEQ ID NO: 420, SEQ ID NO: 429, and SEQ ID NO: 432;
a peptide linker having the amino acid sequence depicted in SEQ ID
NO: 292;
a polypeptide having an amino acid sequence selected from the group
consisting of SEQ ID NO: 38, SEQ ID NO: 48, SEQ ID NO: 58, SEQ ID
NO: 68, SEQ ID NO: 78, SEQ ID NO: 88, SEQ ID NO: 98, SEQ ID NO:
108, SEQ ID NO: 118, SEQ ID NO: 128, SEQ ID NO: 138, SEQ ID NO:
148, SEQ ID NO: 158, SEQ ID NO: 168, SEQ ID NO: 436, SEQ ID NO:
456, SEQ ID NO: 457, SEQ ID NO: 458, SEQ ID NO: 459, SEQ ID NO:
460, SEQ ID NO: 461, SEQ ID NO: 462, SEQ ID NO: 463, SEQ ID NO:
464, SEQ ID NO: 465, SEQ ID NO: 466, SEQ ID NO: 467, SEQ ID NO:
468, SEQ ID NO: 469, SEQ ID NO: 470, and SEQ ID NO: 530, followed
by a serine residue at the C-terminus;
a polypeptide having the amino acid sequence depicted in SEQ ID NO:
331; and
a polypeptide comprising in the following order starting from the
N-terminus:
a polypeptide having an amino acid sequence selected from the group
consisting of SEQ ID NO: 37, SEQ ID NO: 47, SEQ ID NO: 57, SEQ ID
NO: 67, SEQ ID NO: 77, SEQ ID NO: 87, SEQ ID NO: 97, SEQ ID NO:
107, SEQ ID NO: 117, SEQ ID NO: 127, SEQ ID NO: 137, SEQ ID NO:
147, SEQ ID NO: 157, SEQ ID NO: 167, SEQ ID NO: 435, SEQ ID NO:
445, SEQ ID NO: 446, SEQ ID NO: 447, SEQ ID NO: 448, SEQ ID NO:
449, SEQ ID NO: 450, SEQ ID NO: 451, SEQ ID NO: 452, SEQ ID NO:
453, SEQ ID NO: 454, and SEQ ID NO: 455, and SEQ ID NO: 529;
a peptide linker having the amino acid sequence depicted in SEQ ID
NO: 292;
a polypeptide having an amino acid sequence selected from the group
consisting of SEQ ID NO: 349, SEQ ID NO: 358, SEQ ID NO: 367, SEQ
ID NO: 376, SEQ ID NO: 385, SEQ ID NO: 394, SEQ ID NO: 403, SEQ ID
NO: 412, SEQ ID NO: 421, SEQ ID NO: 430, and SEQ ID NO: 433
followed by a serine residue at the C-terminus; and
a polypeptide having the amino acid sequence depicted in SEQ ID NO:
332;
(e) a polypeptide comprising in the following order starting from
the N-terminus:
a polypeptide having an amino acid sequence selected from the group
consisting of SEQ ID NO: 348, SEQ ID NO: 357, SEQ ID NO: 366, SEQ
ID NO: 375, SEQ ID NO: 384, SEQ ID NO: 393, SEQ ID NO: 402, SEQ ID
NO: 411, SEQ ID NO: 420, SEQ ID NO: 429, and SEQ ID NO: 432;
a peptide linker having the amino acid sequence depicted in SEQ ID
NO: 292;
a polypeptide having an amino acid sequence selected from the group
consisting of SEQ ID NO: 38, SEQ ID NO: 48, SEQ ID NO: 58, SEQ ID
NO: 68, SEQ ID NO: 78, SEQ ID NO: 88, SEQ ID NO: 98, SEQ ID NO:
108, SEQ ID NO: 118, SEQ ID NO: 128, SEQ ID NO: 138, SEQ ID NO:
148, SEQ ID NO: 158, SEQ ID NO: 168, SEQ ID NO: 436, SEQ ID NO:
456, SEQ ID NO: 457, SEQ ID NO: 458, SEQ ID NO: 459, SEQ ID NO:
460, SEQ ID NO: 461, SEQ ID NO: 462, SEQ ID NO: 463, SEQ ID NO:
464, SEQ ID NO: 465, SEQ ID NO: 466, SEQ ID NO: 467, SEQ ID NO:
468, SEQ ID NO: 469, SEQ ID NO: 470, and SEQ ID NO: 530; and
a polypeptide having the amino acid sequence depicted in SEQ ID NO:
333; and
a polypeptide comprising in the following order starting from the
N-terminus:
a polypeptide having an amino acid sequence selected from the group
consisting of SEQ ID NO: 37, SEQ ID NO: 47, SEQ ID NO: 57, SEQ ID
NO: 67, SEQ ID NO: 77, SEQ ID NO: 87, SEQ ID NO: 97, SEQ ID NO:
107, SEQ ID NO: 117, SEQ ID NO: 127, SEQ ID NO: 137, SEQ ID NO:
147, SEQ ID NO: 157, and SEQ ID NO: 167, SEQ ID NO: 435, SEQ ID NO:
445, SEQ ID NO: 446, SEQ ID NO: 447, SEQ ID NO: 448, SEQ ID NO:
449, SEQ ID NO: 450, SEQ ID NO: 451, SEQ ID NO: 452, SEQ ID NO:
453, SEQ ID NO: 454, SEQ ID NO: 455, and SEQ ID NO: 529;
a peptide linker having the amino acid sequence depicted in SEQ ID
NO: 292;
a polypeptide having an amino acid sequence selected from the group
consisting of SEQ ID NO: 349, SEQ ID NO: 358, SEQ ID NO: 367, SEQ
ID NO: 376, SEQ ID NO: 385, SEQ ID NO: 394, SEQ ID NO: 403, SEQ ID
NO: 412, SEQ ID NO: 421, SEQ ID NO: 430, and SEQ ID NO: 433
followed by a serine residue at the C-terminus; and
a polypeptide having the amino acid sequence depicted in SEQ ID NO:
334;
(f) a polypeptide comprising in the following order starting from
the N-terminus:
a polypeptide having an amino acid sequence selected from the group
consisting of SEQ ID NO: 39, SEQ ID NO: 49, SEQ ID NO: 59, SEQ ID
NO: 69, SEQ ID NO: 79, SEQ ID NO: 89, SEQ ID NO: 99, SEQ ID NO:
109, SEQ ID NO: 119, SEQ ID NO: 129, SEQ ID NO: 139, SEQ ID NO:
149, SEQ ID NO: 159, SEQ ID NO: 169, SEQ ID NO: 437, SEQ ID NO:
471, SEQ ID NO: 472, SEQ ID NO: 473, SEQ ID NO: 474, SEQ ID NO:
475, SEQ ID NO: 476, SEQ ID NO: 477, SEQ ID NO: 478, SEQ ID NO:
479, SEQ ID NO: 480, SEQ ID NO: 481, SEQ ID NO: 482, SEQ ID NO:
483, SEQ ID NO: 484, SEQ ID NO: 485, SEQ ID NO: 486, SEQ ID NO:
487, SEQ ID NO: 488, SEQ ID NO: 489, SEQ ID NO: 490, SEQ ID NO:
491, SEQ ID NO: 492, SEQ ID NO: 493, and SEQ ID NO: 531;
a peptide linker having an amino acid sequence selected from the
group consisting of SEQ ID NOs: 285-293;
a polypeptide having an amino acid sequence selected from the group
consisting of SEQ ID NO: 350, SEQ ID NO: 359, SEQ ID NO: 368, SEQ
ID NO: 377, SEQ ID NO: 386, SEQ ID NO: 395, SEQ ID NO: 404, SEQ ID
NO: 413, SEQ ID NO: 422, SEQ ID NO: 431, and SEQ ID NO: 434;
a polypeptide having the amino acid sequence depicted in SEQ ID NO:
335; and
a polypeptide having the amino acid sequence depicted in SEQ ID NO:
336;
(g) a polypeptide comprising in the following order starting from
the N-terminus:
a polypeptide having an amino acid sequence selected from the group
consisting of SEQ ID NO: 39, SEQ ID NO: 49, SEQ ID NO: 59, SEQ ID
NO: 69, SEQ ID NO: 79, SEQ ID NO: 89, SEQ ID NO: 99, SEQ ID NO:
109, SEQ ID NO: 119, SEQ ID NO: 129, SEQ ID NO: 139, SEQ ID NO:
149, SEQ ID NO: 159, SEQ ID NO: 169, SEQ ID NO: 437, SEQ ID NO:
471, SEQ ID NO: 472, SEQ ID NO: 473, SEQ ID NO: 474, SEQ ID NO:
475, SEQ ID NO: 476, SEQ ID NO: 477, SEQ ID NO: 478, SEQ ID NO:
479, SEQ ID NO: 480, SEQ ID NO: 481, SEQ ID NO: 482, SEQ ID NO:
483, SEQ ID NO: 484, SEQ ID NO: 485, SEQ ID NO: 486, SEQ ID NO:
487, SEQ ID NO: 488, SEQ ID NO: 489, SEQ ID NO: 490, SEQ ID NO:
491, SEQ ID NO: 492, SEQ ID NO: 493, and SEQ ID NO: 531; and
a polypeptide having the amino acid sequence depicted in SEQ ID NO:
337; and
a polypeptide comprising in the following order starting from the
N-terminus:
a polypeptide having an amino acid sequence selected from the group
consisting of SEQ ID NO: 350, SEQ ID NO: 359, SEQ ID NO: 368, SEQ
ID NO: 377, SEQ ID NO: 386, SEQ ID NO: 395, SEQ ID NO: 404, SEQ ID
NO: 413, SEQ ID NO: 422, SEQ ID NO: 431, and SEQ ID NO: 434;
and
a polypeptide having the amino acid sequence depicted in SEQ ID NO:
338;
(h) a polypeptide comprising in the following order starting from
the N-terminus:
a polypeptide having an amino acid sequence selected from the group
consisting of SEQ ID NO: 39, SEQ ID NO: 49, SEQ ID NO: 59, SEQ ID
NO: 69, SEQ ID NO: 79, SEQ ID NO: 89, SEQ ID NO: 99, SEQ ID NO:
109, SEQ ID NO: 119, SEQ ID NO: 129, SEQ ID NO: 139, SEQ ID NO:
149, SEQ ID NO: 159, SEQ ID NO: 169, SEQ ID NO: 437, SEQ ID NO:
471, SEQ ID NO: 472, SEQ ID NO: 473, SEQ ID NO: 474, SEQ ID NO:
475, SEQ ID NO: 476, SEQ ID NO: 477, SEQ ID NO: 478, SEQ ID NO:
479, SEQ ID NO: 480, SEQ ID NO: 481, SEQ ID NO: 482, SEQ ID NO:
483, SEQ ID NO: 484, SEQ ID NO: 485, SEQ ID NO: 486, SEQ ID NO:
487, SEQ ID NO: 488, SEQ ID NO: 489, SEQ ID NO: 490, SEQ ID NO:
491, SEQ ID NO: 492, SEQ ID NO: 493, and SEQ ID NO: 531; and
a polypeptide having the amino acid sequence depicted in SEQ ID NO:
339; and
a polypeptide comprising in the following order starting from the
N-terminus:
a polypeptide having an amino acid sequence selected from the group
consisting of SEQ ID NO: 350, SEQ ID NO: 359, SEQ ID NO: 368, SEQ
ID NO: 377, SEQ ID NO: 386, SEQ ID NO: 395, SEQ ID NO: 404, SEQ ID
NO: 413, SEQ ID NO: 422, SEQ ID NO: 431, and SEQ ID NO: 434;
and
a polypeptide having the amino acid sequence depicted in SEQ ID NO:
340; or
(i) a polypeptide comprising in the following order starting from
the N-terminus:
a polypeptide having an amino acid sequence selected from the group
consisting of SEQ ID NO: 39, SEQ ID NO: 49, SEQ ID NO: 59, SEQ ID
NO: 69, SEQ ID NO: 79, SEQ ID NO: 89, SEQ ID NO: 99, SEQ ID NO:
109, SEQ ID NO: 119, SEQ ID NO: 129, SEQ ID NO: 139, SEQ ID NO:
149, SEQ ID NO: 159, SEQ ID NO: 169, SEQ ID NO: 437, SEQ ID NO:
471, SEQ ID NO: 472, SEQ ID NO: 473, SEQ ID NO: 474, SEQ ID NO:
475, SEQ ID NO: 476, SEQ ID NO: 477, SEQ ID NO: 478, SEQ ID NO:
479, SEQ ID NO: 480, SEQ ID NO: 481, SEQ ID NO: 482, SEQ ID NO:
483, SEQ ID NO: 484, SEQ ID NO: 485, SEQ ID NO: 486, SEQ ID NO:
487, SEQ ID NO: 488, SEQ ID NO: 489, SEQ ID NO: 490, SEQ ID NO:
491, SEQ ID NO: 492, SEQ ID NO: 493, and SEQ ID NO: 531;
a peptide linker having an amino acid sequence selected from the
group consisting of SEQ ID NOs: 285-293;
a polypeptide having an amino acid sequence selected from the group
consisting of SEQ ID NO: 350, SEQ ID NO: 359, SEQ ID NO: 368, SEQ
ID NO: 377, SEQ ID NO: 386, SEQ ID NO: 395, SEQ ID NO: 404, SEQ ID
NO: 413, SEQ ID NO: 422, SEQ ID NO: 431, and SEQ ID NO: 434;
and
a polypeptide having the amino acid sequence depicted in SEQ ID NO:
341.
For example, a preferred bispecific antibody construct of the
present invention comprises or consists of a polypeptide selected
from the group consisting of those depicted in: SEQ ID NO: 224, SEQ
ID NO: 225, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID
NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO:
233, SEQ ID NO: 234, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO:
237; SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244, SEQ ID NO:
245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO:
249, SEQ ID NO: 250, and SEQ ID NO: 251.
As described above, several preferred antibody constructs of the
invention are modified by fusion with another moiety such as
albumin or albumin variants. If these fusion constructs are
characterized for their properties, such as in particular their
target affinity or cytotoxic activity, the skilled person will be
aware that similar fusion constructs or unmodified bispecific
antibody constructs can be expected to have similar (or possibly
even better) properties. For example, if a bispecific antibody
construct fused with albumin has an appreciable or desirable
cytotoxic activity or target affinity, it can be expected that the
same/similar or even a higher cytotoxic activity/target affinity
will be observed for the same construct w/o albumin.
According to another preferred embodiment, the bispecific antibody
construct of the invention comprises (in addition to the two
binding domains) a third domain which comprises two polypeptide
monomers, each comprising a hinge, a CH2 and a CH3 domain, wherein
said two polypeptides (or polypeptide monomers) are fused to each
other via a peptide linker. Preferably, said third domain comprises
in an N- to C-terminal order: hinge-CH2-CH3-linker-hinge-CH2-CH3.
Preferred amino acid sequences for said third domain are depicted
in SEQ ID NOs: 541-548. Each of said polypeptide monomers
preferably has an amino acid sequence that is selected from the
group consisting of SEQ ID NOs: 533-540, or that is at least 90%
identical to those sequences. In another preferred embodiment, the
first and second binding domains of the bispecific antibody
construct of the invention are fused to the third domain via a
peptide linker which is for example selected from the group
consisting of SEQ ID NOs: 285, 286, 288, 289, 290, 292 and 293.
In line with the present invention, a "hinge" is an IgG hinge
region. This region can be identified by analogy using the Kabat
numbering, see Kabat positions 223-243. In line with the above, the
minimal requirement for a "hinge" are the amino acid residues
corresponding to the IgG1 sequence stretch of D231 to P243
according to the Kabat numbering. The terms CH2 and CH3 refer to
the immunoglobulin heavy chain constant regions 2 and 3. These
regions can as well be identified by analogy using the Kabat
numbering, see Kabat positions 244-360 for CH2 and Kabat positions
361-478 for CH3. Is is understood that there is some variation
between the immunoglobulins in terms of their IgG1 Fc region, IgG2
Fc region, IgG3 Fc region, IgG4 Fc region, IgM Fc region, IgA Fc
region, IgD Fc region and IgE Fc region (see, e.g., Padlan,
Molecular Immunology, 31(3), 169-217 (1993)). The term Fc monomer
refers to the last two heavy chain constant regions of IgA, IgD,
and IgG, and the last three heavy chain constant regions of IgE and
IgM. The Fc monomer can also include the flexible hinge N-terminal
to these domains. For IgA and IgM, the Fc monomer may include the J
chain. For IgG, the Fc portion comprises immunoglobulin domains CH2
and CH3 and the hinge between the first two domains and CH2.
Although the boundaries of the Fc portion of an immunoglobulin may
vary, an example for a human IgG heavy chain Fc portion comprising
a functional hinge, CH2 and CH3 domain can be defined e.g. to
comprise residues D231 (of the hinge domain) to P476 (of the
C-terminus of the CH3 domain), or D231 to L476, respectively, for
IgG4, wherein the numbering is according to Kabat.
The antibody construct of the invention may hence comprise in an N-
to C-terminal order:
(a) the first binding domain;
(b) a peptide linker having an amino acid sequence selected from
the group consisting of SEQ ID NOs: 286, 292 and 293;
(c) the second binding domain;
(d) a peptide linker having an amino acid sequence selected from
the group consisting of SEQ ID NOs: 285, 286, 288, 289, 290, 292
and 293;
(e) the first polypeptide monomer of the third domain (comprising a
hinge, a CH2 and a CH3 domain);
(f) a peptide linker having an amino acid sequence selected from
the group consisting of SEQ ID NOs: 550, 551, 552 and 553; and
(g) the second polypeptide monomer of the third domain (comprising
a hinge, a CH2 and a CH3 domain).
It is also preferred that the antibody construct of the invention
comprises in an N- to C-terminal order:
the first binding domain having an amino acid sequence selected
from the group consisting of SEQ ID NO: 39, SEQ ID NO: 49, SEQ ID
NO: 59, SEQ ID NO: 69, SEQ ID NO: 79, SEQ ID NO: 89, SEQ ID NO: 99,
SEQ ID NO: 109, SEQ ID NO: 119, SEQ ID NO: 129, SEQ ID NO: 139, SEQ
ID NO: 149, SEQ ID NO: 159, SEQ ID NO: 169, SEQ ID NO: 437, SEQ ID
NO: 471, SEQ ID NO: 472, SEQ ID NO: 473, SEQ ID NO: 474, SEQ ID NO:
475, SEQ ID NO: 476, SEQ ID NO: 477, SEQ ID NO: 478, SEQ ID NO:
479, SEQ ID NO: 480, SEQ ID NO: 481, SEQ ID NO: 482, SEQ ID NO:
483, SEQ ID NO: 484, SEQ ID NO: 485, SEQ ID NO: 486, SEQ ID NO:
487, SEQ ID NO: 488, SEQ ID NO: 489, SEQ ID NO: 490, SEQ ID NO:
491, SEQ ID NO: 492, SEQ ID NO: 493, and SEQ ID NO: 531;
a peptide linker having an amino acid sequence selected from the
group consisting of SEQ ID NOs: 286, 292 and 293;
the second binding domain having an amino acid sequence selected
from the group consisting of SEQ ID NO: 350, SEQ ID NO: 359, SEQ ID
NO: 368, SEQ ID NO: 377, SEQ ID NO: 386, SEQ ID NO: 395, SEQ ID NO:
404, SEQ ID NO: 413, SEQ ID NO: 422, SEQ ID NO: 431, and SEQ ID NO:
434;
a peptide linker having an amino acid sequence selected from the
group consisting of SEQ ID NOs: 285, 286, 288, 289, 290, 292 and
293; and
the third domain having an amino acid sequence selected from the
group consisting of SEQ ID NOs: 541-548.
Hence, in a preferred embodiment, the antibody construct of the
present invention comprises or consists of a polypeptide selected
from the group consisting of those depicted in SEQ ID NO: 517, SEQ
ID NO: 518, SEQ ID NO: 519, SEQ ID NO: 520, SEQ ID NO: 521, SEQ ID
NO: 522, SEQ ID NO: 523, SEQ ID NO: 524, SEQ ID NO: 525, SEQ ID NO:
526, SEQ ID NO: 527, and SEQ ID NO: 528.
The sequence table (Table 18) also provides sequence variations of
the binders denominated DLL3-4 and DLL3-14. The point mutations
that were inserted into these sequence variants are identified
according to the position of this mutation within the respective
scFv molecule. It is understood that an alternative way of
identifying these positions is also possible, depending on the
polypeptide of reference, which could as well be the CDR region or
the VH/VL region. For example, the variant denominated DLL3-4-001
has a G44C-G243C double mutation in its scFv molecule (SEQ ID NO:
437). This translates into a G44C mutation in the corresponding VH
chain (SEQ ID NO: 435) and a G101C mutation in the corresponding VL
chain (SEQ ID NO: 436).
Covalent modifications of the antibody constructs are also included
within the scope of this invention, and are generally, but not
always, done post-translationally. For example, several types of
covalent modifications of the antibody construct are introduced
into the molecule by reacting specific amino acid residues of the
antibody construct with an organic derivatizing agent that is
capable of reacting with selected side chains or the N- or
C-terminal residues.
Cysteinyl residues most commonly are reacted with
.alpha.-haloacetates (and corresponding amines), such as
chloroacetic acid or chloroacetamide, to give carboxymethyl or
carboxyamidomethyl derivatives. Cysteinyl residues also are
derivatized by reaction with bromotrifluoroacetone,
.alpha.-bromo-.beta.-(5-imidozoyl)propionic acid, chloroacetyl
phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl
2-pyridyl disulfide, p-chloromercuribenzoate,
2-chloromercuri-4-nitrophenol, or
chloro-7-nitrobenzo-2-oxa-1,3-diazole.
Histidyl residues are derivatized by reaction with
diethylpyrocarbonate at pH 5.5-7.0 because this agent is relatively
specific for the histidyl side chain. Para-bromophenacyl bromide
also is useful; the reaction is preferably performed in 0.1 M
sodium cacodylate at pH 6.0. Lysinyl and amino terminal residues
are reacted with succinic or other carboxylic acid anhydrides.
Derivatization with these agents has the effect of reversing the
charge of the lysinyl residues. Other suitable reagents for
derivatizing alpha-amino-containing residues include imidoesters
such as methyl picolinimidate; pyridoxal phosphate; pyridoxal;
chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea;
2,4-pentanedione; and transaminase-catalyzed reaction with
glyoxylate.
Arginyl residues are modified by reaction with one or several
conventional reagents, among them phenylglyoxal, 2,3-butanedione,
1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine
residues requires that the reaction be performed in alkaline
conditions because of the high pKa of the guanidine functional
group. Furthermore, these reagents may react with the groups of
lysine as well as the arginine epsilon-amino group.
The specific modification of tyrosyl residues may be made, with
particular interest in introducing spectral labels into tyrosyl
residues by reaction with aromatic diazonium compounds or
tetranitromethane. Most commonly, N-acetylimidizole and
tetranitromethane are used to form 0-acetyl tyrosyl species and
3-nitro derivatives, respectively. Tyrosyl residues are iodinated
using .sup.125I or .sup.131I to prepare labeled proteins for use in
radioimmunoassay, the chloramine T method described above being
suitable.
Carboxyl side groups (aspartyl or glutamyl) are selectively
modified by reaction with carbodiimides (R'--N.dbd.C.dbd.N--R'),
where R and R' are optionally different alkyl groups, such as
1-cyclohexyl-3-(2-morpholinyl-4-ethyl) carbodiimide or
1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore,
aspartyl and glutamyl residues are converted to asparaginyl and
glutaminyl residues by reaction with ammonium ions.
Derivatization with bifunctional agents is useful for crosslinking
the antibody constructs of the present invention to a
water-insoluble support matrix or surface for use in a variety of
methods. Commonly used crosslinking agents include, e.g.,
1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,
N-hydroxysuccinimide esters, for example, esters with
4-azidosalicylic acid, homobifunctional imidoesters, including
disuccinimidyl esters such as
3,3'-dithiobis(succinimidylpropionate), and bifunctional maleimides
such as bis-N-maleimido-1,8-octane. Derivatizing agents such as
methyl-3-[(p-azidophenyl)dithio]propioimidate yield
photoactivatable intermediates that are capable of forming
crosslinks in the presence of light. Alternatively, reactive
water-insoluble matrices such as cyanogen bromide-activated
carbohydrates and the reactive substrates as described in U.S. Pat.
Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and
4,330,440 are employed for protein immobilization.
Glutaminyl and asparaginyl residues are frequently deamidated to
the corresponding glutamyl and aspartyl residues, respectively.
Alternatively, these residues are deamidated under mildly acidic
conditions. Either form of these residues falls within the scope of
this invention.
Other modifications include hydroxylation of proline and lysine,
phosphorylation of hydroxyl groups of seryl or threonyl residues,
methylation of the .alpha.-amino groups of lysine, arginine, and
histidine side chains (T. E. Creighton, Proteins: Structure and
Molecular Properties, W. H. Freeman & Co., San Francisco, 1983,
pp. 79-86), acetylation of the N-terminal amine, and amidation of
any C-terminal carboxyl group.
Another type of covalent modification of the antibody constructs
included within the scope of this invention comprises altering the
glycosylation pattern of the protein. As is known in the art,
glycosylation patterns can depend on both the sequence of the
protein (e.g., the presence or absence of particular glycosylation
amino acid residues, discussed below), or the host cell or organism
in which the protein is produced. Particular expression systems are
discussed below.
Glycosylation of polypeptides is typically either N-linked or
O-linked. N-linked refers to the attachment of the carbohydrate
moiety to the side chain of an asparagine residue. The tri-peptide
sequences asparagine-X-serine and asparagine-X-threonine, where X
is any amino acid except proline, are the recognition sequences for
enzymatic attachment of the carbohydrate moiety to the asparagine
side chain. Thus, the presence of either of these tri-peptide
sequences in a polypeptide creates a potential glycosylation site.
O-linked glycosylation refers to the attachment of one of the
sugars N-acetylgalactosamine, galactose, or xylose, to a
hydroxyamino acid, most commonly serine or threonine, although
5-hydroxyproline or 5-hydroxylysine may also be used.
Addition of glycosylation sites to the antibody construct is
conveniently accomplished by altering the amino acid sequence such
that it contains one or more of the above-described tri-peptide
sequences (for N-linked glycosylation sites). The alteration may
also be made by the addition of, or substitution by, one or more
serine or threonine residues to the starting sequence (for O-linked
glycosylation sites). For ease, the amino acid sequence of an
antibody construct is preferably altered through changes at the DNA
level, particularly by mutating the DNA encoding the polypeptide at
preselected bases such that codons are generated that will
translate into the desired amino acids.
Another means of increasing the number of carbohydrate moieties on
the antibody construct is by chemical or enzymatic coupling of
glycosides to the protein. These procedures are advantageous in
that they do not require production of the protein in a host cell
that has glycosylation capabilities for N- and O-linked
glycosylation. Depending on the coupling mode used, the sugar(s)
may be attached to (a) arginine and histidine, (b) free carboxyl
groups, (c) free sulfhydryl groups such as those of cysteine, (d)
free hydroxyl groups such as those of serine, threonine, or
hydroxyproline, (e) aromatic residues such as those of
phenylalanine, tyrosine, or tryptophan, or (f) the amide group of
glutamine. These methods are described in WO 87/05330, and in Aplin
and Wriston, 1981, CRC Crit. Rev. Biochem., pp. 259-306.
Removal of carbohydrate moieties present on the starting antibody
construct may be accomplished chemically or enzymatically. Chemical
deglycosylation requires exposure of the protein to the compound
trifluoromethanesulfonic acid, or an equivalent compound. This
treatment results in the cleavage of most or all sugars except the
linking sugar (N-acetylglucosamine or N-acetylgalactosamine), while
leaving the polypeptide intact. Chemical deglycosylation is
described by Hakimuddin et al., 1987, Arch. Biochem. Biophys.
259:52 and by Edge et al., 1981, Anal. Biochem. 118:131. Enzymatic
cleavage of carbohydrate moieties on polypeptides can be achieved
by the use of a variety of endo- and exo-glycosidases as described
by Thotakura et al., 1987, Meth. Enzymol. 138:350. Glycosylation at
potential glycosylation sites may be prevented by the use of the
compound tunicamycin as described by Duskin et al., 1982, J. Biol.
Chem. 257:3105. Tunicamycin blocks the formation of
protein-N-glycoside linkages.
Other modifications of the antibody construct are also contemplated
herein. For example, another type of covalent modification of the
antibody construct comprises linking the antibody construct to
various non-proteinaceous polymers, including, but not limited to,
various polyols such as polyethylene glycol, polypropylene glycol,
polyoxyalkylenes, or copolymers of polyethylene glycol and
polypropylene glycol, in the manner set forth in U.S. Pat. Nos.
4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.
In addition, as is known in the art, amino acid substitutions may
be made in various positions within the antibody construct, e.g. in
order to facilitate the addition of polymers such as PEG.
In some embodiments, the covalent modification of the antibody
constructs of the invention comprises the addition of one or more
labels. The labelling group may be coupled to the antibody
construct via spacer arms of various lengths to reduce potential
steric hindrance. Various methods for labelling proteins are known
in the art and can be used in performing the present invention. The
term "label" or "labelling group" refers to any detectable label.
In general, labels fall into a variety of classes, depending on the
assay in which they are to be detected--the following examples
include, but are not limited to:
isotopic labels, which may be radioactive or heavy isotopes, such
as radioisotopes or radionuclides (e.g., 3H, 14C, 15N, 35S, 89Zr,
90Y, 99Tc, 111In, 125I, 131I)
magnetic labels (e.g., magnetic particles)
redox active moieties
optical dyes (including, but not limited to, chromophores,
phosphors and fluorophores) such as fluorescent groups (e.g., FITC,
rhodamine, lanthanide phosphors), chemiluminescent groups, and
fluorophores which can be either "small molecule" fluores or
proteinaceous fluores
enzymatic groups (e.g. horseradish peroxidase,
.beta.-galactosidase, luciferase, alkaline phosphatase)
biotinylated groups
predetermined polypeptide epitopes recognized by a secondary
reporter (e.g., leucine zipper pair sequences, binding sites for
secondary antibodies, metal binding domains, epitope tags,
etc.)
By "fluorescent label" is meant any molecule that may be detected
via its inherent fluorescent properties. Suitable fluorescent
labels include, but are not limited to, fluorescein, rhodamine,
tetramethylrhodamine, eosin, erythrosin, coumarin,
methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow,
Cascade BlueJ, Texas Red, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy
5, Cy 5.5, LC Red 705, Oregon green, the Alexa-Fluor dyes (Alexa
Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 546, Alexa
Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660, Alexa
Fluor 680), Cascade Blue, Cascade Yellow and R-phycoerythrin (PE)
(Molecular Probes, Eugene, Oreg.), FITC, Rhodamine, and Texas Red
(Pierce, Rockford, Ill.), Cy5, Cy5.5, Cy7 (Amersham Life Science,
Pittsburgh, Pa.). Suitable optical dyes, including fluorophores,
are described in Molecular Probes Handbook by Richard P.
Haugland.
Suitable proteinaceous fluorescent labels also include, but are not
limited to, green fluorescent protein, including a Renilla,
Ptilosarcus, or Aequorea species of GFP (Chalfie et al., 1994,
Science 263:802-805), EGFP (Clontech Laboratories, Inc., Genbank
Accession Number U55762), blue fluorescent protein (BFP, Quantum
Biotechnologies, Inc. 1801 de Maisonneuve Blvd. West, 8th Floor,
Montreal, Quebec, Canada H3H 1J9; Stauber, 1998, Biotechniques
24:462-471; Heim et al., 1996, Curr. Biol. 6:178-182), enhanced
yellow fluorescent protein (EYFP, Clontech Laboratories, Inc.),
luciferase (Ichiki et al., 1993, J. Immunol. 150:5408-5417), .beta.
galactosidase (Nolan et al., 1988, Proc. Natl. Acad. Sci. U.S.A.
85:2603-2607) and Renilla (WO92/15673, WO95/07463, WO98/14605,
WO98/26277, WO99/49019, U.S. Pat. Nos. 5,292,658; 5,418,155;
5,683,888; 5,741,668; 5,777,079; 5,804,387; 5,874,304; 5,876,995;
5,925,558).
Leucine zipper domains are peptides that promote oligomerization of
the proteins in which they are found. Leucine zippers were
originally identified in several DNA-binding proteins (Landschulz
et al., 1988, Science 240:1759), and have since been found in a
variety of different proteins. Among the known leucine zippers are
naturally occurring peptides and derivatives thereof that dimerize
or trimerize. Examples of leucine zipper domains suitable for
producing soluble oligomeric proteins are described in PCT
application WO 94/10308, and the leucine zipper derived from lung
surfactant protein D (SPD) described in Hoppe et al., 1994, FEBS
Letters 344:191. The use of a modified leucine zipper that allows
for stable trimerization of a heterologous protein fused thereto is
described in Fanslow et al., 1994, Semin. Immunol. 6:267-78. In one
approach, recombinant fusion proteins comprising a DLL3 antibody
fragment or derivative fused to a leucine zipper peptide are
expressed in suitable host cells, and the soluble oligomeric DLL3
antibody fragments or derivatives that form are recovered from the
culture supernatant.
The antibody construct of the invention may also comprise
additional domains, which are e.g. helpful in the isolation of the
molecule or relate to an adapted pharmacokinetic profile of the
molecule. Domains helpful for the isolation of an antibody
construct may be selected from peptide motives or secondarily
introduced moieties, which can be captured in an isolation method,
e.g. an isolation column. Non-limiting embodiments of such
additional domains comprise peptide motives known as Myc-tag,
HAT-tag, HA-tag, TAP-tag, GST-tag, chitin binding domain (CBD-tag),
maltose binding protein (MBP-tag), Flag-tag, Strep-tag and variants
thereof (e.g. StrepII-tag) and His-tag. All herein disclosed
antibody constructs characterized by the identified CDRs are
preferred to comprise a His-tag domain, which is generally known as
a repeat of consecutive His residues in the amino acid sequence of
a molecule, preferably of five, and more preferably of six His
residues (hexa-histidine). The His-tag may be located e.g. at the
N- or C-terminus of the antibody construct, preferably it is
located at the C-terminus. Most preferably, a hexa-histidine tag
(HHHHHH) is linked via peptide bond to the C-terminus of the
antibody construct according to the invention.
The first binding domain of the antibody construct of the present
invention binds to human DLL3 on the surface of a target cell. The
preferred amino acid sequence of human DLL3 is represented by SEQ
ID NO: 252. It is understood that the term "on the surface", in the
context of the present invention, means that the binding domain
specifically binds to an epitope comprised within the DLL3
extracellular domain (DLL3 ECD). The first binding domain according
to the invention hence preferably binds to DLL3 when it is
expressed by naturally expressing cells or cell lines, and/or by
cells or cell lines transformed or (stably/transiently) transfected
with DLL3. In a preferred embodiment the first binding domain also
binds to DLL3 when DLL3 is used as a "target" or "ligand" molecule
in an in vitro binding assay such as BIAcore or Scatchard. The
"target cell" can be any prokaryotic or eukaryotic cell expressing
DLL3 on its surface; preferably the target cell is a cell that is
part of the human or animal body, such as a specific DLL3
expressing cancer or tumor cell.
The term "DLL3 ECD" refers to a form of DLL3 which is essentially
free of transmembrane and cytoplasmic domains of DLL3. It will be
understood by the skilled artisan that the transmembrane domain
identified for the DLL3 polypeptide of the present invention is
identified pursuant to criteria routinely employed in the art for
identifying that type of hydrophobic domain. The exact boundaries
of a transmembrane domain may vary but most likely by no more than
about 5 amino acids at either end of the domain specifically
mentioned herein. A preferred human DLL3 ECD is shown in SEQ ID NO:
253.
The affinity of the first binding domain for human DLL3 is
preferably .ltoreq.20 nM, more preferably .ltoreq.10 nM, even more
preferably .ltoreq.5 nM, even more preferably .ltoreq.2 nM, even
more preferably .ltoreq.1 nM, even more preferably .ltoreq.0.6 nM,
even more preferably .ltoreq.0.5 nM, and most preferably
.ltoreq.0.4 nM. The affinity can be measured for example in a
BIAcore assay or in a Scatchard assay, e.g. as described in the
Examples. Other methods of determining the affinity are also
well-known to the skilled person.
T cells or T lymphocytes are a type of lymphocyte (itself a type of
white blood cell) that play a central role in cell-mediated
immunity. There are several subsets of T cells, each with a
distinct function. T cells can be distinguished from other
lymphocytes, such as B cells and NK cells, by the presence of a T
cell receptor (TCR) on the cell surface. The TCR is responsible for
recognizing antigens bound to major histocompatibility complex
(MHC) molecules and is composed of two different protein chains. In
95% of the T cells, the TCR consists of an alpha (a) and beta
(.beta.) chain. When the TCR engages with antigenic peptide and MHC
(peptide/MHC complex), the T lymphocyte is activated through a
series of biochemical events mediated by associated enzymes,
co-receptors, specialized adaptor molecules, and activated or
released transcription factors.
The CD3 receptor complex is a protein complex and is composed of
four chains. In mammals, the complex contains a CD3.gamma. (gamma)
chain, a CD3.delta. (delta) chain, and two CD3.epsilon. (epsilon)
chains. These chains associate with the T cell receptor (TCR) and
the so-called (zeta) chain to form the T cell receptor CD3 complex
and to generate an activation signal in T lymphocytes. The
CD3.gamma. (gamma), CD3.delta. (delta), and CD3.epsilon. (epsilon)
chains are highly related cell-surface proteins of the
immunoglobulin superfamily containing a single extracellular
immunoglobulin domain. The intracellular tails of the CD3 molecules
contain a single conserved motif known as an immunoreceptor
tyrosine-based activation motif or ITAM for short, which is
essential for the signaling capacity of the TCR. The CD3 epsilon
molecule is a polypeptide which in humans is encoded by the CD3E
gene which resides on chromosome 11. The most preferred epitope of
CD3 epsilon is comprised within amino acid residues 1-27 of the
human CD3 epsilon extracellular domain.
The redirected lysis of target cells via the recruitment of T cells
by a multispecific, at least bispecific, antibody construct
involves cytolytic synapse formation and delivery of perforin and
granzymes. The engaged T cells are capable of serial target cell
lysis, and are not affected by immune escape mechanisms interfering
with peptide antigen processing and presentation, or clonal T cell
differentiation; see, for example, WO 2007/042261.
Cytotoxicity mediated by DLL3.times.CD3 bispecific antibody
constructs can be measured in various ways. See Examples 8.1 to
8.7. Effector cells can be e.g. stimulated enriched (human) CD8
positive T cells or unstimulated (human) peripheral blood
mononuclear cells (PBMC). If the target cells are of macaque origin
or express or are transfected with macaque DLL3, the effector cells
should also be of macaque origin such as a macaque T cell line,
e.g. 4119LnPx. The target cells should express (at least the
extracellular domain of) DLL3, e.g. human or macaque DLL3. Target
cells can be a cell line (such as CHO) which is stably or
transiently transfected with DLL3, e.g. human or macaque DLL3.
Alternatively, the target cells can be a DLL3 positive natural
expresser cell line, such as the human lung carcinoma cell line
SHP-77. Usually EC50 values are expected to be lower with target
cell lines expressing higher levels of DLL3 on the cell surface.
The effector to target cell (E:T) ratio is usually about 10:1, but
can also vary. Cytotoxic activity of DLL3.times.CD3 bispecific
antibody constructs can be measured in a 51-chromium release assay
(incubation time of about 18 hours) or in a in a FACS-based
cytotoxicity assay (incubation time of about 48 hours).
Modifications of the assay incubation time (cytotoxic reaction) are
also possible. Other methods of measuring cytotoxicity are
well-known to the skilled person and comprise MTT or MTS assays,
ATP-based assays including bioluminescent assays, the
sulforhodamine B (SRB) assay, WST assay, clonogenic assay and the
ECIS technology.
The cytotoxic activity mediated by DLL3.times.CD3 bispecific
antibody constructs of the present invention is preferably measured
in a cell-based cytotoxicity assay. It may also be measured in a
51-chromium release assay. It is represented by the EC50 value,
which corresponds to the half maximal effective concentration
(concentration of the antibody construct which induces a cytotoxic
response halfway between the baseline and maximum). Preferably, the
EC50 value of the DLL3.times.CD3 bispecific antibody constructs is
.ltoreq.5000 pM or .ltoreq.4000 pM, more preferably
.ltoreq..ltoreq.3000 pM or .ltoreq..ltoreq.2000 pM, even more
preferably .ltoreq.1000 pM or .ltoreq.500 pM, even more preferably
.ltoreq.400 pM or .ltoreq.300 pM, even more preferably .ltoreq.200
pM, even more preferably .ltoreq.100 pM, even more preferably
.ltoreq.50 pM, even more preferably .ltoreq.20 pM or .ltoreq.10 pM,
and most preferably .ltoreq.5 pM.
The above given EC50 values can be measured in different assays.
The skilled person is aware that an EC50 value can be expected to
be lower when stimulated/enriched CD8+ T cells are used as effector
cells, compared with unstimulated PBMC. It can furthermore be
expected that the EC50 values are lower when the target cells
express a high number of the target antigen compared with a low
target expression rat. For example, when stimulated/enriched human
CD8+ T cells are used as effector cells (and either DLL3
transfected cells such as CHO cells or a DLL3 positive human lung
carcinoma cell line SHP-77 are used as target cells), the EC50
value of the DLL3 xCD3 bispecific antibody construct is preferably
.ltoreq.1000 pM, more preferably .ltoreq.500 pM, even more
preferably .ltoreq.250 pM, even more preferably .ltoreq.100 pM,
even more preferably .ltoreq.50 pM, even more preferably .ltoreq.10
pM, and most preferably .ltoreq.5 pM. When human PBMCs are used as
effector cells, the EC50 value of the DLL3.times.CD3 bispecific
antibody construct is preferably .ltoreq.5000 pM or .ltoreq.4000 pM
(in particular when the target cells are a DLL3 positive human lung
carcinoma cell line SHP-77), more preferably .ltoreq.2000 pM (in
particular when the target cells are DLL3 transfected cells such as
CHO cells), more preferably .ltoreq.1000 pM or .ltoreq.500 pM, even
more preferably .ltoreq.200 pM, even more preferably .ltoreq.150
pM, even more preferably .ltoreq.100 pM, and most preferably
.ltoreq.50 pM, or lower. When a macaque T cell line such as
LnPx4119 is used as effector cells, and a macaque DLL3 transfected
cell line such as CHO cells is used as target cell line, the EC50
value of the DLL3.times.CD3 bispecific antibody construct is
preferably .ltoreq.2000 pM or .ltoreq.1500 pM, more preferably
.ltoreq.1000 pM or .ltoreq.500 pM, even more preferably .ltoreq.300
pM or .ltoreq.250 pM, even more preferably .ltoreq.100 pM, and most
preferably .ltoreq.50 pM.
Preferably, the DLL3.times.CD3 bispecific antibody constructs of
the present invention do not induce/mediate lysis or do not
essentially induce/mediate lysis of DLL3 negative cells such as CHO
cells. The term "do not induce lysis", "do not essentially induce
lysis", "do not mediate lysis" or "do not essentially mediate
lysis" means that an antibody construct of the present invention
does not induce or mediate lysis of more than 30%, preferably not
more than 20%, more preferably not more than 10%, particularly
preferably not more than 9%, 8%, 7%, 6% or 5% of DLL3 negative
cells, whereby lysis of a DLL3 positive human lung carcinoma cell
line SHP-77 (see above) is set to be 100%. This usually applies for
concentrations of the antibody construct of up to 500 nM. The
skilled person knows how to measure cell lysis without further ado.
Moreover, the present specification teaches specific instructions
how to measure cell lysis.
The difference in cytotoxic activity between the monomeric and the
dimeric isoform of individual DLL3.times.CD3 bispecific antibody
constructs is referred to as "potency gap". This potency gap can
e.g. be calculated as ratio between EC50 values of the molecule's
monomeric and dimeric form, see Example 15. Potency gaps of the
DLL3.times.CD3 bispecific antibody constructs of the present
invention are preferably .ltoreq.5, more preferably .ltoreq.4, even
more preferably .ltoreq.3, even more preferably .ltoreq.2 and most
preferably .ltoreq.1.
The first and/or the second (or any further) binding domain(s) of
the antibody construct of the invention is/are preferably
cross-species specific for members of the mammalian order of
primates. Cross-species specific CD3 binding domains are, for
example, described in WO 2008/119567. According to one embodiment,
the first and/or second binding domain, in addition to binding to
human DLL3 and human CD3, respectively, will also bind to DLL3/CD3
of primates including (but not limited to) new world primates (such
as Callithrix jacchus, Saguinus oedipus or Saimiri sciureus), old
world primates (such as baboons and macaques), gibbons, orangutans
and non-human homininae. It is envisaged that the first binding
domain of the antibody construct of the invention which binds to
human DLL3 on the surface of a target cell also binds at least to
macaque DLL3, and/or the second binding domain which binds to human
CD3 on the surface of a T cell also binds at least to macaque CD3.
A preferred macaque is Macaca fascicularis. Macaca mulatta (Rhesus)
is also envisaged.
A preferred bispecific antibody construct of the invention
comprises a first binding domain which binds to human DLL3 on the
surface of a target cell and a second binding domain which binds to
human CD3 on the surface of a T cell and at least macaque CD3. In
one aspect of this embodiment, the first binding domain binds to an
epitope of DLL3 which is comprised within the region as depicted in
SEQ ID NO: 260.
In one aspect of the invention, the first binding domain binds to
human DLL3 and further binds to macaque DLL3, such as DLL3 of
Macaca fascicularis, and more preferably, to macaque DLL3 ECD. A
preferred Macaca fascicularis DLL3 is depicted in SEQ ID NO: 271. A
preferred macaque DLL3 ECD is depicted in SEQ ID NO: 272. The
affinity of the first binding domain for macaque DLL3 is preferably
.ltoreq.15 nM, more preferably .ltoreq.0 nM, even more preferably
.ltoreq.5 nM, even more preferably .ltoreq.1 nM, even more
preferably .ltoreq.0.5 nM, even more preferably .ltoreq.0.1 nM, and
most preferably .ltoreq.0.05 nM or even .ltoreq.0.01 nM.
Preferably the affinity gap of the antibody constructs according to
the invention for binding macaque DLL3 versus human DLL3 [ma
DLL3:hu DLL3] (as determined e.g. by BiaCore or by Scatchard
analysis) is between 0.1 and 10, more preferably between 0.2 and 5,
even more preferably between 0.3 and 4, even more preferably
between 0.5 and 3 or between 0.5 and 2.5, and most preferably
between 0.5 and 2 or between 0.6 and 2. See Examples 3 and 4.
In one embodiment of the antibody construct of the invention, the
second binding domain binds to human CD3 epsilon and to Callithrix
jacchus, Saguinus oedipus or Saimiri sciureus CD3 epsilon.
Preferably, the second binding domain binds to an extracellular
epitope of these CD3 epsilon chains. It is also envisaged that the
second binding domain binds to an extracellular epitope of the
human and the Macaca CD3 epsilon chain. The most preferred epitope
of CD3 epsilon is comprised within amino acid residues 1-27 of the
human CD3 epsilon extracellular domain. Even more specifically, the
epitope comprises at least the amino acid sequence
Gln-Asp-Gly-Asn-Glu. Callithrix jacchus and Saguinus oedipus are
both new world primate belonging to the family of Callitrichidae,
while Saimiri sciureus is a new world primate belonging to the
family of Cebidae.
It is particularly preferred for the antibody construct of the
present invention that the second binding domain which binds to
human CD3 on the surface of a T cell comprises a VL region
comprising CDR-L1, CDR-L2 and CDR-L3 selected from:
(a) CDR-L1 as depicted in SEQ ID NO: 27 of WO 2008/119567, CDR-L2
as depicted in SEQ ID NO: 28 of WO 2008/119567 and CDR-L3 as
depicted in SEQ ID NO: 29 of WO 2008/119567;
(b) CDR-L1 as depicted in SEQ ID NO: 117 of WO 2008/119567, CDR-L2
as depicted in SEQ ID NO: 118 of WO 2008/119567 and CDR-L3 as
depicted in SEQ ID NO: 119 of WO 2008/119567; and
(c) CDR-L1 as depicted in SEQ ID NO: 153 of WO 2008/119567, CDR-L2
as depicted in SEQ ID NO: 154 of WO 2008/119567 and CDR-L3 as
depicted in SEQ ID NO: 155 of WO 2008/119567.
In an alternatively preferred embodiment of the antibody construct
of the present invention, the second binding domain which binds to
human CD3 on the surface of a T cell comprises a VH region
comprising CDR-H 1, CDR-H2 and CDR-H3 selected from:
(a) CDR-H1 as depicted in SEQ ID NO: 12 of WO 2008/119567, CDR-H2
as depicted in SEQ ID NO: 13 of WO 2008/119567 and CDR-H3 as
depicted in SEQ ID NO: 14 of WO 2008/119567;
(b) CDR-H1 as depicted in SEQ ID NO: 30 of WO 2008/119567, CDR-H2
as depicted in SEQ ID NO: 31 of WO 2008/119567 and CDR-H3 as
depicted in SEQ ID NO: 32 of WO 2008/119567;
(c) CDR-H1 as depicted in SEQ ID NO: 48 of WO 2008/119567, CDR-H2
as depicted in SEQ ID NO: 49 of WO 2008/119567 and CDR-H3 as
depicted in SEQ ID NO: 50 of WO 2008/119567;
(d) CDR-H1 as depicted in SEQ ID NO: 66 of WO 2008/119567, CDR-H2
as depicted in SEQ ID NO: 67 of WO 2008/119567 and CDR-H3 as
depicted in SEQ ID NO: 68 of WO 2008/119567;
(e) CDR-H1 as depicted in SEQ ID NO: 84 of WO 2008/119567, CDR-H2
as depicted in SEQ ID NO: 85 of WO 2008/119567 and CDR-H3 as
depicted in SEQ ID NO: 86 of WO 2008/119567;
(f) CDR-H1 as depicted in SEQ ID NO: 102 of WO 2008/119567, CDR-H2
as depicted in SEQ ID NO: 103 of WO 2008/119567 and CDR-H3 as
depicted in SEQ ID NO: 104 of WO 2008/119567;
(g) CDR-H1 as depicted in SEQ ID NO: 120 of WO 2008/119567, CDR-H2
as depicted in SEQ ID NO: 121 of WO 2008/119567 and CDR-H3 as
depicted in SEQ ID NO: 122 of WO 2008/119567;
(h) CDR-H1 as depicted in SEQ ID NO: 138 of WO 2008/119567, CDR-H2
as depicted in SEQ ID NO: 139 of WO 2008/119567 and CDR-H3 as
depicted in SEQ ID NO: 140 of WO 2008/119567;
(i) CDR-H1 as depicted in SEQ ID NO: 156 of WO 2008/119567, CDR-H2
as depicted in SEQ ID NO: 157 of WO 2008/119567 and CDR-H3 as
depicted in SEQ ID NO: 158 of WO 2008/119567; and
(j) CDR-H1 as depicted in SEQ ID NO: 174 of WO 2008/119567, CDR-H2
as depicted in SEQ ID NO: 175 of WO 2008/119567 and CDR-H3 as
depicted in SEQ ID NO: 176 of WO 2008/119567.
It is further preferred for the antibody construct of the present
invention that the second binding domain which binds to human CD3
on the surface of a T cell comprises a VL region selected from the
group consisting of a VL region as depicted in SEQ ID NO: 35, 39,
125, 129, 161 or 165 of WO 2008/119567.
It is alternatively preferred that the second binding domain which
binds to human CD3 on the surface of a T cell comprises a VH region
selected from the group consisting of a VH region as depicted in
SEQ ID NO: 15, 19, 33, 37, 51, 55, 69, 73, 87, 91, 105, 109, 123,
127, 141, 145, 159, 163, 177 or 181 of WO 2008/119567.
More preferably, the antibody construct of the present invention is
characterized by the second binding domain which binds to human CD3
on the surface of a T cell comprising a VL region and a VH region
selected from the group consisting of:
(a) a VL region as depicted in SEQ ID NO: 17 or 21 of WO
2008/119567 and a VH region as depicted in SEQ ID NO: 15 or 19 of
WO 2008/119567;
(b) a VL region as depicted in SEQ ID NO: 35 or 39 of WO
2008/119567 and a VH region as depicted in SEQ ID NO: 33 or 37 of
WO 2008/119567;
(c) a VL region as depicted in SEQ ID NO: 53 or 57 of WO
2008/119567 and a VH region as depicted in SEQ ID NO: 51 or 55 of
WO 2008/119567;
(d) a VL region as depicted in SEQ ID NO: 71 or 75 of WO
2008/119567 and a VH region as depicted in SEQ ID NO: 69 or 73 of
WO 2008/119567;
(e) a VL region as depicted in SEQ ID NO: 89 or 93 of WO
2008/119567 and a VH region as depicted in SEQ ID NO: 87 or 91 of
WO 2008/119567;
(f) a VL region as depicted in SEQ ID NO: 107 or 111 of WO
2008/119567 and a VH region as depicted in SEQ ID NO: 105 or 109 of
WO 2008/119567;
(g) a VL region as depicted in SEQ ID NO: 125 or 129 of WO
2008/119567 and a VH region as depicted in SEQ ID NO: 123 or 127 of
WO 2008/119567;
(h) a VL region as depicted in SEQ ID NO: 143 or 147 of WO
2008/119567 and a VH region as depicted in SEQ ID NO: 141 or 145 of
WO 2008/119567;
(i) a VL region as depicted in SEQ ID NO: 161 or 165 of WO
2008/119567 and a VH region as depicted in SEQ ID NO: 159 or 163 of
WO 2008/119567; and
(j) a VL region as depicted in SEQ ID NO: 179 or 183 of WO
2008/119567 and a VH region as depicted in SEQ ID NO: 177 or 181 of
WO 2008/119567.
According to a preferred embodiment of the antibody construct of
the present invention, the binding domains and in particular the
second binding domain (which binds to human CD3 on the surface of a
T cell) have the following format: The pairs of VH regions and VL
regions are in the format of a single chain antibody (scFv). The VH
and VL regions are arranged in the order VH-VL or VL-VH. It is
preferred that the VH-region is positioned N-terminally of a linker
sequence, and the VL-region is positioned C-terminally of the
linker sequence.
A preferred embodiment of the above described antibody construct of
the present invention is characterized by the second binding domain
which binds to human CD3 on the surface of a T cell comprising an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 23, 25, 41, 43, 59, 61, 77, 79, 95, 97, 113, 115, 131, 133,
149, 151, 167, 169, 185 or 187 of WO 2008/119567.
Hence, in one embodiment, the antibody construct of the invention
comprises a polypeptide selected from the group consisting of those
depicted in SEQ ID NO: 40, SEQ ID NO: 50, SEQ ID NO: 60, SEQ ID NO:
70, SEQ ID NO: 80, SEQ ID NO: 90, SEQ ID NO: 100, SEQ ID NO: 110,
SEQ ID NO: 120, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ
ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID
NO: 438 and SEQ ID NO: 532. These antibody constructs have a first
binding domain which binds to an epitope of DLL3 which is comprised
within the region as depicted in SEQ ID NO: 258.
In an alternative embodiment, the antibody construct of the
invention comprises a polypeptide selected from the group
consisting of those depicted in SEQ ID NO: 130, SEQ ID NO: 140, SEQ
ID NO: 150, SEQ ID NO: 160, SEQ ID NO: 170; SEQ ID NO: 218, SEQ ID
NO: 219, SEQ ID NO: 220, SEQ ID NO: 494, SEQ ID NO: 495, SEQ ID NO:
496, SEQ ID NO: 497, SEQ ID NO: 498, SEQ ID NO: 499, SEQ ID NO:
500, SEQ ID NO: 501, SEQ ID NO: 502, SEQ ID NO: 503, SEQ ID NO:
504, SEQ ID NO: 505, SEQ ID NO: 506, SEQ ID NO: 507, SEQ ID NO:
508, SEQ ID NO: 509, SEQ ID NO: 510, SEQ ID NO: 511, SEQ ID NO:
512, SEQ ID NO: 513, SEQ ID NO: 514, SEQ ID NO: 515, and SEQ ID NO:
516. These antibody constructs have a first binding domain which
binds to an epitope of DLL3 which is comprised within the region as
depicted in SEQ ID NO: 259.
Amino acid sequence modifications of the antibody constructs
described herein are also contemplated. For example, it may be
desirable to improve the binding affinity and/or other biological
properties of the antibody construct. Amino acid sequence variants
of the antibody constructs are prepared by introducing appropriate
nucleotide changes into the antibody constructs nucleic acid, or by
peptide synthesis. All of the below described amino acd sequence
modifications should result in an antibody construct which still
retains the desired biological activity (binding to DLL3 and to
CD3) of the unmodified parental molecule.
The term "amino acid" or "amino acid residue" typically refers to
an amino acid having its art recognized definition such as an amino
acid selected from the group consisting of: alanine (Ala or A);
arginine (Arg or R); asparagine (Asn or N); aspartic acid (Asp or
D); cysteine (Cys or C); glutamine (GIn or Q); glutamic acid (Glu
or E); glycine (Gly or G); histidine (His or H); isoleucine (He or
I): leucine (Leu or L); lysine (Lys or K); methionine (Met or M);
phenylalanine (Phe or F); pro line (Pro or P); serine (Ser or S);
threonine (Thr or T); tryptophan (Trp or W); tyrosine (Tyr or Y);
and valine (Val or V), although modified, synthetic, or rare amino
acids may be used as desired. Generally, amino acids can be grouped
as having a nonpolar side chain (e.g., Ala, Cys, He, Leu, Met, Phe,
Pro, Val); a negatively charged side chain (e.g., Asp, Glu); a
positively charged sidechain (e.g., Arg, His, Lys); or an uncharged
polar side chain (e.g., Asn, Cys, Gin, Gly, His, Met, Phe, Ser,
Thr, Trp, and Tyr).
Amino acid modifications include, for example, deletions from,
and/or insertions into, and/or substitutions of, residues within
the amino acid sequences of the antibody constructs. Any
combination of deletion, insertion, and substitution is made to
arrive at the final construct, provided that the final construct
possesses the desired characteristics. The amino acid changes also
may alter post-translational processes of the antibody constructs,
such as changing the number or position of glycosylation sites.
For example, 1, 2, 3, 4, 5, or 6 amino acids may be inserted or
deleted in each of the CDRs (of course, dependent on their length),
while 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, or 25 amino acids may be inserted or deleted in each of
the FRs. Preferably, amino acid sequence insertions include amino-
and/or carboxyl-terminal fusions ranging in length from 1, 2, 3, 4,
5, 6, 7, 8, 9 or 10 residues to polypeptides containing a hundred
or more residues, as well as intra-sequence insertions of single or
multiple amino acid residues. An insertional variant of the
antibody construct of the invention includes the fusion to the
N-terminus or to the C-terminus of the antibody construct of an
enzyme or the fusion to a polypeptide which increases the serum
half-life of the antibody construct.
The sites of greatest interest for substitutional mutagenesis
include the CDRs of the heavy and/or light chain, in particular the
hypervariable regions, but FR alterations in the heavy and/or light
chain are also contemplated. The substitutions are preferably
conservative substitutions as described herein. Preferably, 1, 2,
3, 4, 5, 6, 7, 8, 9, or 10 amino acids may be substituted in a CDR,
while 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, or 25 amino acids may be substituted in the framework
regions (FRs), depending on the length of the CDR or FR. For
example, if a CDR sequence encompasses 6 amino acids, it is
envisaged that one, two or three of these amino acids are
substituted. Similarly, if a CDR sequence encompasses 15 amino
acids it is envisaged that one, two, three, four, five or six of
these amino acids are substituted.
A useful method for identification of certain residues or regions
of the antibody constructs that are preferred locations for
mutagenesis is called "alanine scanning mutagenesis" as described
by Cunningham and Wells in Science, 244: 1081-1085 (1989). Here, a
residue or group of target residues within the antibody construct
is/are identified (e.g. charged residues such as arg, asp, his,
lys, and glu) and replaced by a neutral or negatively charged amino
acid (most preferably alanine or polyalanine) to affect the
interaction of the amino acids with the epitope.
Those amino acid locations demonstrating functional sensitivity to
the substitutions are then refined by introducing further or other
variants at, or for, the sites of substitution. Thus, while the
site or region for introducing an amino acid sequence variation is
predetermined, the nature of the mutation per se needs not to be
predetermined. For example, to analyze or optimize the performance
of a mutation at a given site, alanine scanning or random
mutagenesis may be conducted at a target codon or region, and the
expressed antibody construct variants are screened for the optimal
combination of desired activity. Techniques for making substitution
mutations at predetermined sites in the DNA having a known sequence
are well known, for example, M13 primer mutagenesis and PCR
mutagenesis. Screening of the mutants is done using assays of
antigen binding activities, such as DLL3 or CD3 binding.
Generally, if amino acids are substituted in one or more or all of
the CDRs of the heavy and/or light chain, it is preferred that the
then-obtained "substituted" sequence is at least 60% or 65%, more
preferably 70% or 75%, even more preferably 80% or 85%, and
particularly preferably 90% or 95% identical to the "original" CDR
sequence. This means that it is dependent of the length of the CDR
to which degree it is identical to the "substituted" sequence. For
example, a CDR having 5 amino acids is preferably 80% identical to
its substituted sequence in order to have at least one amino acid
substituted. Accordingly, the CDRs of the antibody construct may
have different degrees of identity to their substituted sequences,
e.g., CDRL1 may have 80%, while CDRL3 may have 90%.
Preferred substitutions (or replacements) are conservative
substitutions. However, any substitution (including
non-conservative substitution or one or more from the "exemplary
substitutions" listed in Table 1, below) is envisaged as long as
the antibody construct retains its capability to bind to DLL3 via
the first binding domain and to CD3 or CD3 epsilon via the second
binding domain and/or its CDRs have an identity to the then
substituted sequence (at least 60% or 65%, more preferably 70% or
75%, even more preferably 80% or 85%, and particularly preferably
90% or 95% identical to the "original" CDR sequence).
Conservative substitutions are shown in Table 1 under the heading
of "preferred substitutions". If such substitutions result in a
change in biological activity, then more substantial changes,
denominated "exemplary substitutions" in Table 1, or as further
described below in reference to amino acid classes, may be
introduced and the products screened for a desired
characteristic.
TABLE-US-00001 TABLE 1 Amino acid substitutions Original Exemplary
Substitutions Preferred Substitutions Ala (A) val, leu, ile val Arg
(R) lys, gln, asn lys Asn (N) gln, his, asp, lys, arg gln Asp (D)
glu, asn glu Cys (C) ser, ala ser Gln (Q) asn, glu asn Glu (E) asp,
gln asp Gly (G) Ala ala His (H) asn, gln, lys, arg arg Ile (I) leu,
val, met, ala, phe leu Leu (L) norleucine, ile, val, met, ala ile
Lys (K) arg, gln, asn arg Met (M) leu, phe, ile leu Phe (F) leu,
val, ile, ala, tyr tyr Pro (P) Ala ala Ser (S) Thr thr Thr (T) Ser
ser Trp (W) tyr, phe tyr Tyr (Y) trp, phe, thr, ser phe Val (V)
ile, leu, met, phe, ala leu
Substantial modifications in the biological properties of the
antibody construct of the present invention are accomplished by
selecting substitutions that differ significantly in their effect
on maintaining (a) the structure of the polypeptide backbone in the
area of the substitution, for example, as a sheet or helical
conformation, (b) the charge or hydrophobicity of the molecule at
the target site, or (c) the bulk of the side chain. Naturally
occurring residues are divided into groups based on common
side-chain properties: (1) hydrophobic: norleucine, met, ala, val,
leu, ile; (2) neutral hydrophilic: cys, ser, thr; (3) acidic: asp,
glu; (4) basic: asn, gin, his, lys, arg; (5) residues that
influence chain orientation: gly, pro; and (6) aromatic: trp, tyr,
phe.
Non-conservative substitutions will entail exchanging a member of
one of these classes for another class. Any cysteine residue not
involved in maintaining the proper conformation of the antibody
construct may be substituted, generally with serine, to improve the
oxidative stability of the molecule and prevent aberrant
crosslinking. Conversely, cysteine bond(s) may be added to the
antibody to improve its stability (particularly where the antibody
is an antibody fragment such as an Fv fragment).
For amino acid sequences, sequence identity and/or similarity is
determined by using standard techniques known in the art,
including, but not limited to, the local sequence identity
algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482, the
sequence identity alignment algorithm of Needleman and Wunsch,
1970, J. Mol. Biol. 48:443, the search for similarity method of
Pearson and Lipman, 1988, Proc. Nat. Acad. Sci. U.S.A. 85:2444,
computerized implementations of these algorithms (GAP, BESTFIT,
FASTA, and TFASTA in the Wisconsin Genetics Software Package,
Genetics Computer Group, 575 Science Drive, Madison, Wis.), the
Best Fit sequence program described by Devereux et al., 1984, Nucl.
Acid Res. 12:387-395, preferably using the default settings, or by
inspection. Preferably, percent identity is calculated by FastDB
based upon the following parameters: mismatch penalty of 1; gap
penalty of 1; gap size penalty of 0.33; and joining penalty of 30,
"Current Methods in Sequence Comparison and Analysis,"
Macromolecule Sequencing and Synthesis, Selected Methods and
Applications, pp 127-149 (1988), Alan R. Liss, Inc.
An example of a useful algorithm is PILEUP. PILEUP creates a
multiple sequence alignment from a group of related sequences using
progressive, pairwise alignments. It can also plot a tree showing
the clustering relationships used to create the alignment. PILEUP
uses a simplification of the progressive alignment method of Feng
& Doolittle, 1987, J. Mol. Evol. 35:351-360; the method is
similar to that described by Higgins and Sharp, 1989, CABIOS
5:151-153. Useful PILEUP parameters including a default gap weight
of 3.00, a default gap length weight of 0.10, and weighted end
gaps.
Another example of a useful algorithm is the BLAST algorithm,
described in: Altschul et al., 1990, J. Mol. Biol. 215:403-410;
Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402; and Karin
et al., 1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873-5787. A
particularly useful BLAST program is the WU-BLAST-2 program which
was obtained from Altschul et al., 1996, Methods in Enzymology
266:460-480. WU-BLAST-2 uses several search parameters, most of
which are set to the default values. The adjustable parameters are
set with the following values: overlap span=1, overlap
fraction=0.125, word threshold (T)=II. The HSP S and HSP S2
parameters are dynamic values and are established by the program
itself depending upon the composition of the particular sequence
and composition of the particular database against which the
sequence of interest is being searched; however, the values may be
adjusted to increase sensitivity.
An additional useful algorithm is gapped BLAST as reported by
Altschul et al., 1993, Nucl. Acids Res. 25:3389-3402. Gapped BLAST
uses BLOSUM-62 substitution scores; threshold T parameter set to 9;
the two-hit method to trigger ungapped extensions, charges gap
lengths of k a cost of 10+k; Xu set to 16, and Xg set to 40 for
database search stage and to 67 for the output stage of the
algorithms. Gapped alignments are triggered by a score
corresponding to about 22 bits.
Generally, the amino acid homology, similarity, or identity between
individual variant CDRs are at least 60% to the sequences depicted
herein, and more typically with preferably increasing homologies or
identities of at least 65% or 70%, more preferably at least 75% or
80%, even more preferably at least 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99%, and almost 100%. In a similar manner,
"percent (%) nucleic acid sequence identity" with respect to the
nucleic acid sequence of the binding proteins identified herein is
defined as the percentage of nucleotide residues in a candidate
sequence that are identical with the nucleotide residues in the
coding sequence of the antibody construct. A specific method
utilizes the BLASTN module of WU-BLAST-2 set to the default
parameters, with overlap span and overlap fraction set to 1 and
0.125, respectively.
Generally, the nucleic acid sequence homology, similarity, or
identity between the nucleotide sequences encoding individual
variant CDRs and the nucleotide sequences depicted herein are at
least 60%, and more typically with preferably increasing homologies
or identities of at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99%, and almost 100%. Thus, a "variant CDR" is one with the
specified homology, similarity, or identity to the parent CDR of
the invention, and shares biological function, including, but not
limited to, at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% of the specificity and/or activity of the parent
CDR.
In one embodiment, the percentage of identity to human germline of
the antibody constructs according to the invention is .gtoreq.70%
or .gtoreq.75%, more preferably .gtoreq.80% or .gtoreq.85%, even
more preferably .gtoreq.90%, and most preferably .gtoreq.91%,
.gtoreq.92%, .gtoreq.93%, .gtoreq.94%, .gtoreq.95% or even
.gtoreq.96%. See Example 7. Identity to human antibody germline
gene products is thought to be an important feature to reduce the
risk of therapeutic proteins to elicit an immune response against
the drug in the patient during treatment. Hwang & Foote
("lImmunogenicity of engineered antibodies"; Methods 36 (2005)
3-10) demonstrate that the reduction of non-human portions of drug
antibody constructs leads to a decrease of risk to induce anti-drug
antibodies in the patients during treatment. By comparing an
exhaustive number of clinically evaluated antibody drugs and the
respective immunogenicity data, the trend is shown that
humanization of the V-regions of antibodies makes the protein less
immunogenic (average 5.1% of patients) than antibodies carrying
unaltered non-human V regions (average 23.59% of patients). A
higher degree of identity to human sequences is hence desirable for
V-region based protein therapeutics in the form of antibody
constructs. For this purpose of determining the germline identity,
the V-regions of VL can be aligned with the amino acid sequences of
human germline V segments and J segments (http colon-slash-slash
vbase.mrc-cpe.cam.ac.uk/) using Vector NTI software and the amino
acid sequence calculated by dividing the identical amino acid
residues by the total number of amino acid residues of the VL in
percent. The same can be for the VH segments (http
colon-slash-slash vbase.mrc-cpc.cam.ac.uk/) with the exception that
the VH CDR3 may be excluded due to its high diversity and a lack of
existing human germline VH CDR3 alignment partners. Recombinant
techniques can then be used to increase sequence identity to human
antibody germline genes.
In a further embodiment, the bispecific antibody constructs of the
present invention exhibit high monomer yields under standard
research scale conditions, e.g., in a standard two-step
purification process. Preferably the monomer yield of the antibody
constructs according to the invention is .gtoreq.0.25 mg/L
supernatant, more preferably .gtoreq.0.5 mg/L, even more preferably
.gtoreq.1 mg/L, and most preferably .gtoreq.3 mg/L supernatant.
Likewise, the yield of the dimeric antibody construct isoforms and
hence the monomer percentage (i.e., monomer: (monomer+dimer)) of
the antibody constructs can be determined. The productivity of
monomeric and dimeric antibody constructs and the calculated
monomer percentage can e.g. be obtained in the SEC purification
step of culture supernatant from standardized research-scale
production in roller bottles. In one embodiment, the monomer
percentage of the antibody constructs is .gtoreq.80%, more
preferably .gtoreq.85%, even more preferably .gtoreq.90%, and most
preferably .gtoreq.95%.
In one embodiment, the antibody constructs have a preferred plasma
stability (ratio of EC50 with plasma to EC50 w/o plasma) of
.ltoreq.5 or .ltoreq.4, more preferably .ltoreq.3.5 or .ltoreq.3,
even more preferably .ltoreq.2.5 or .ltoreq.2, and most preferably
.ltoreq.1.5 or .ltoreq.1. The plasma stability of an antibody
construct can be tested by incubation of the construct in human
plasma at 37.degree. C. for 24 hours followed by EC50 determination
in a 51-chromium release cytotoxicity assay. The effector cells in
the cytotoxicity assay can be stimulated enriched human CD8
positive T cells. Target cells can e.g. be CHO cells transfected
with human DLL3. The effector to target cell (E:T) ratio can be
chosen as 10:1. The human plasma pool used for this purpose is
derived from the blood of healthy donors collected by EDTA coated
syringes. Cellular components are removed by centrifugation and the
upper plasma phase is collected and subsequently pooled. As
control, antibody constructs are diluted immediately prior to the
cytotoxicity assay in RPMI-1640 medium. The plasma stability is
calculated as ratio of EC50 (after plasma incubation) to EC50
(control). See Example 11.
It is furthermore preferred that the monomer to dimer conversion of
antibody constructs of the invention is low. The conversion can be
measured under different conditions and analyzed by high
performance size exclusion chromatography. See Example 9. For
example, incubation of the monomeric isoforms of the antibody
constructs can be carried out for 7 days at 37.degree. C. and
concentrations of e.g. 100 .mu.g/ml or 250 .mu.g/ml in an
incubator. Under these conditions, it is preferred that the
antibody constructs of the invention show a dimer percentage that
is .ltoreq.5%, more preferably .ltoreq.4%, even more preferably
.ltoreq.3%, even more preferably .ltoreq.2.5%, even more preferably
.ltoreq.2%, even more preferably .ltoreq.1.5%, and most preferably
.ltoreq.1% or .ltoreq.0.5% or even 0%.
It is also preferred that the bispecific antibody constructs of the
present invention present with very low dimer conversion after a
number of freeze/thaw cycles. For example, the antibody construct
monomer is adjusted to a concentration of 250 .mu.g/ml e.g. in
generic formulation buffer and subjected to three freeze/thaw
cycles (freezing at -80.degree. C. for 30 min followed by thawing
for 30 min at room temperature), followed by high performance SEC
to determine the percentage of initially monomeric antibody
construct, which had been converted into dimeric antibody
construct. Preferably the dimer percentages of the bispecific
antibody constructs are .ltoreq.5%, more preferably .ltoreq.4%,
even more preferably .ltoreq.3%, even more preferably .ltoreq.2.5%,
even more preferably .ltoreq.2%, even more preferably .ltoreq.1.5%,
and most preferably .ltoreq.1% or even 50.5%, for example after
three freeze/thaw cycles.
The bispecific antibody constructs of the present invention
preferably show a favorable thermostability with aggregation
temperatures .gtoreq.45.degree. C. or .gtoreq.50.degree. C., more
preferably .gtoreq.52.degree. C. or .gtoreq.54.degree. C., even
more preferably .gtoreq.56.degree. C. or .gtoreq.57.degree. C., and
most preferably .gtoreq.58.degree. C. or .gtoreq.59.degree. C. The
thermostability parameter can be determined in terms of antibody
aggregation temperature as follows: Antibody solution at a
concentration 250 .mu.g/ml is transferred into a single use cuvette
and placed in a Dynamic Light Scattering (DLS) device. The sample
is heated from 40.degree. C. to 70.degree. C. at a heating rate of
0.5.degree. C./min with constant acquisition of the measured
radius. Increase of radius indicating melting of the protein and
aggregation is used to calculate the aggregation temperature of the
antibody. See Example 10.
Alternatively, temperature melting curves can be determined by
Differential Scanning Calorimetry (DSC) to determine intrinsic
biophysical protein stabilities of the antibody constructs. These
experiments are performed using a MicroCal LLC (Northampton, Mass.,
U.S.A) VP-DSC device. The energy uptake of a sample containing an
antibody construct is recorded from 20.degree. C. to 90.degree. C.
compared to a sample containing only the formulation buffer. The
antibody constructs are adjusted to a final concentration of 250
.mu.g/ml e.g. in SEC running buffer. For recording of the
respective melting curve, the overall sample temperature is
increased stepwise. At each temperature T energy uptake of the
sample and the formulation buffer reference is recorded. The
difference in energy uptake Cp (kcal/mole/.degree. C.) of the
sample minus the reference is plotted against the respective
temperature. The melting temperature is defined as the temperature
at the first maximum of energy uptake.
It is furthermore envisaged that the DLL3.times.CD3 bispecific
antibody constructs of the invention do not cross-react with (i.e.,
do not essentially bind to) the human DLL3 paralogues DLL1 and/or
DLL4. Furthermore, it is envisaged that the DLL3.times.CD3
bispecific antibody constructs of the invention do not cross-react
with (i.e., do not essentially bind to) the macaque/cyno DLL3
paralogues DLL1 and/or DLL4. See Example 6.
The DLL3.times.CD3 bispecific antibody constructs of the invention
are also envisaged to have a turbidity (as measured by OD340 after
concentration of purified monomeric antibody construct to 2.5 mg/ml
and over night incubation) of .ltoreq.0.2, preferably of
.ltoreq.0.15, more preferably of .ltoreq.0.12, even more preferably
of .ltoreq.0.1, and most preferably of .ltoreq.0.08. See Example
12.
The DLL3.times.CD3 bispecific antibody constructs of the invention
are also envisaged to not be internalized or to not undergo
significant internalization by the target cell. The rate of
internalization can be assayed e.g. as described in Example 16.
Preferably, the internalization rate (e.g. measured as a decrease
in cytotoxicity) is .ltoreq.20% after a 2 hour (pre-)incubation of
the antibody construct with the target cell, more preferably
.ltoreq.15%, even more preferably .ltoreq.10%, and most preferably
.ltoreq.5%.
It is furthermore envisaged that shed or soluble DLL3 does not
significantly impair the efficacy or biologic activity of the
DLL3.times.CD3 bispecific antibody constructs of the invention.
This can be measured e.g. in a cytotoxicity assay where soluble
DLL3 is added at increasing concentrations to the assay, e.g. at 0
nM-0.3 nM-0.7 nM-1 nM-3 nM-7 nM-12 nM. The EC50 value of the tested
antibody construct should not be significantly increased in the
presence of soluble DLL3. See Example 17.
In a further embodiment the antibody construct according to the
invention is stable at acidic pH. The more tolerant the antibody
construct behaves at unphysiologic pH such as pH 5.5 (a pH which is
required to run e.g. a cation exchange chromatography), the higher
is the recovery of the antibody construct eluted from an ion
exchange column relative to the total amount of loaded protein.
Recovery of the antibody construct from an ion (e.g., cation)
exchange column at pH 5.5 is preferably .gtoreq.30%, more
preferably .gtoreq.40%, more preferably .gtoreq.50%, even more
preferably .gtoreq.60%, even more preferably .gtoreq.70%, even more
preferably .gtoreq.80%, even more preferably .gtoreq.90%, even more
preferably .gtoreq.95%, and most preferably .gtoreq.99%. See
Example 13.
It is furthermore envisaged that the bispecific antibody constructs
of the present invention exhibit therapeutic efficacy or anti-tumor
activity. This can e.g. be assessed in a study as disclosed in the
following example of an advanced stage human tumor xenograft
model:
On day 1 of the study, 5.times.10.sup.6 cells of a human DLL3
positive cancer cell line (e.g. SHP-77) are subcutaneously injected
in the right dorsal flank of female NOD/SCID mice. When the mean
tumor volume reaches about 100 mm.sup.3, in vitro expanded human
CD3 positive T cells are transplanted into the mice by injection of
about 2.times.10.sup.7 cells into the peritoneal cavity of the
animals. Mice of vehicle control group 1 do not receive effector
cells and are used as an untransplanted control for comparison with
vehicle control group 2 (receiving effector cells) to monitor the
impact of T cells alone on tumor growth. The antibody treatment
starts when the mean tumor volume reaches about 200 mm.sup.3. The
mean tumor size of each treatment group on the day of treatment
start should not be statistically different from any other group
(analysis of variance). Mice are treated with 0.5 mg/kg/day of a
DLL3.times.CD3 bispecifc antibody construct by intravenous bolus
injection for about 15 to 20 days. Tumors are measured by caliper
during the study and progress evaluated by intergroup comparison of
tumor volumes (TV). The tumor growth inhibition T/C [%] is
determined by calculating TV as T/C %=100.times. (median TV of
analyzed group)/(median TV of control group 2).
The skilled person knows how to modify or adapt certain parameters
of this study, such as the number of injected tumor cells, the site
of injection, the number of transplanted human T cells, the amount
of bispecific antibody constructs to be administered, and the
timelines, while still arriving at a meaningful and reproducible
result. Preferably, the tumor growth inhibition T/C [%] is
.ltoreq.70 or .ltoreq.60, more preferably .ltoreq.50 or .ltoreq.40,
even more preferably .ltoreq.30 or .ltoreq.20 and most preferably
.ltoreq.10 or .ltoreq.5 or even .ltoreq.2.5.
The invention further provides a polynucleotide/nucleic acid
molecule encoding an antibody construct of the invention.
A polynucleotide is a biopolymer composed of 13 or more nucleotide
monomers covalently bonded in a chain. DNA (such as cDNA) and RNA
(such as mRNA) are examples of polynucleotides with distinct
biological function. Nucleotides are organic molecules that serve
as the monomers or subunits of nucleic acid molecules like DNA or
RNA. The nucleic acid molecule or polynucleotide can be double
stranded and single stranded, linear and circular. It is preferably
comprised in a vector which is preferably comprised in a host cell.
Said host cell is, e.g. after transformation or transfection with
the vector or the polynucleotide of the invention, capable of
expressing the antibody construct. For that purpose the
polynucleotide or nucleic acid molecule is operatively linked with
control sequences.
The genetic code is the set of rules by which information encoded
within genetic material (nucleic acids) is translated into
proteins. Biological decoding in living cells is accomplished by
the ribosome which links amino acids in an order specified by mRNA,
using tRNA molecules to carry amino acids and to read the mRNA
three nucleotides at a time. The code defines how sequences of
these nucleotide triplets, called codons, specify which amino acid
will be added next during protein synthesis. With some exceptions,
a three-nucleotide codon in a nucleic acid sequence specifies a
single amino acid. Because the vast majority of genes are encoded
with exactly the same code, this particular code is often referred
to as the canonical or standard genetic code. While the genetic
code determines the protein sequence for a given coding region,
other genomic regions can influence when and where these proteins
are produced.
Furthermore, the invention provides a vector comprising a
polynucleotide/nucleic acid molecule of the invention.
A vector is a nucleic acid molecule used as a vehicle to transfer
(foreign) genetic material into a cell. The term "vector"
encompasses--but is not restricted to--plasmids, viruses, cosmids
and artificial chromosomes. In general, engineered vectors comprise
an origin of replication, a multicloning site and a selectable
marker. The vector itself is generally a nucleotide sequence,
commonly a DNA sequence, that comprises an insert (transgene) and a
larger sequence that serves as the "backbone" of the vector. Modern
vectors may encompass additional features besides the transgene
insert and a backbone: promoter, genetic marker, antibiotic
resistance, reporter gene, targeting sequence, protein purification
tag. Vectors called expression vectors (expression constructs)
specifically are for the expression of the transgene in the target
cell, and generally have control sequences.
The term "control sequences" refers to DNA sequences necessary for
the expression of an operably linked coding sequence in a
particular host organism. The control sequences that are suitable
for prokaryotes, for example, include a promoter, optionally an
operator sequence, and a ribosome binding site. Eukaryotic cells
are known to utilize promoters, polyadenylation signals, and
enhancers.
A nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
example, DNA for a presequence or secretory leader is operably
linked to DNA for a polypeptide if it is expressed as a preprotein
that participates in the secretion of the polypeptide; a promoter
or enhancer is operably linked to a coding sequence if it affects
the transcription of the sequence; or a ribosome binding site is
operably linked to a coding sequence if it is positioned so as to
facilitate translation. Generally, "operably linked" means that the
DNA sequences being linked are contiguous, and, in the case of a
secretory leader, contiguous and in reading phase. However,
enhancers do not have to be contiguous. Linking is accomplished by
ligation at convenient restriction sites. If such sites do not
exist, the synthetic oligonucleotide adaptors or linkers are used
in accordance with conventional practice.
"Transfection" is the process of deliberately introducing nucleic
acid molecules or polynucleotides (including vectors) into target
cells. The term is mostly used for non-viral methods in eukaryotic
cells. Transduction is often used to describe virus-mediated
transfer of nucleic acid molecules or polynucleotides. Transfection
of animal cells typically involves opening transient pores or
"holes" in the cell membrane, to allow the uptake of material.
Transfection can be carried out using calcium phosphate, by
electroporation, by cell squeezing or by mixing a cationic lipid
with the material to produce liposomes, which fuse with the cell
membrane and deposit their cargo inside.
The term "transformation" is used to describe non-viral transfer of
nucleic acid molecules or polynucleotides (including vectors) into
bacteria, and also into non-animal eukaryotic cells, including
plant cells. Transformation is hence the genetic alteration of a
bacterial or non-animal eukaryotic cell resulting from the direct
uptake through the cell membrane(s) from its surroundings and
subsequent incorporation of exogenous genetic material (nucleic
acid molecules). Transformation can be effected by artificial
means. For transformation to happen, cells or bacteria must be in a
state of competence, which might occur as a time-limited response
to environmental conditions such as starvation and cell
density.
Moreover, the invention provides a host cell transformed or
transfected with the polynucleotide/nucleic acid molecule or with
the vector of the invention.
As used herein, the terms "host cell" or "recipient cell" are
intended to include any individual cell or cell culture that can be
or has/have been recipients of vectors, exogenous nucleic acid
molecules, and polynucleotides encoding the antibody construct of
the present invention; and/or recipients of the antibody construct
itself. The introduction of the respective material into the cell
is carried out by way of transformation, transfection and the like.
The term "host cell" is also intended to include progeny or
potential progeny of a single cell. Because certain modifications
may occur in succeeding generations due to either natural,
accidental, or deliberate mutation or due to environmental
influences, such progeny may not, in fact, be completely identical
(in morphology or in genomic or total DNA complement) to the parent
cell, but is still included within the scope of the term as used
herein. Suitable host cells include prokaryotic or eukaryotic
cells, and also include but are not limited to bacteria, yeast
cells, fungi cells, plant cells, and animal cells such as insect
cells and mammalian cells, e.g., murine, rat, macaque or human.
The antibody construct of the invention can be produced in
bacteria. After expression, the antibody construct of the invention
is isolated from the E. coli cell paste in a soluble fraction and
can be purified through, e.g., affinity chromatography and/or size
exclusion. Final purification can be carried out similar to the
process for purifying antibody expressed e.g., in CHO cells.
In addition to prokaryotes, eukaryotic microbes such as filamentous
fungi or yeast are suitable cloning or expression hosts for the
antibody construct of the invention. Saccharomyces cerevisiae, or
common baker's yeast, is the most commonly used among lower
eukaryotic host microorganisms. However, a number of other genera,
species, and strains are commonly available and useful herein, such
as Schizosaccharomyces pombe, Kluyveromyces hosts such as K.
lactis, K. fragilis (ATCC 12424), K. bulgaricus (ATCC 16045), K.
wickeramii (ATCC 24178), K. waltii (ATCC 56500), K. drosophilarum
(ATCC 36906), K. thermotolerans, and K. marxianus; yarrowia (EP 402
226); Pichia pastoris (EP 183 070); Candida; Trichoderma reesia (EP
244 234); Neurospora crassa; Schwanniomyces such as Schwanniomyces
occidentalis; and filamentous fungi such as Neurospora,
Penicillium, Tolypocladium, and Aspergillus hosts such as A.
nidulans and A. niger.
Suitable host cells for the expression of glycosylated antibody
construct of the invention are derived from multicellular
organisms. Examples of invertebrate cells include plant and insect
cells. Numerous baculoviral strains and variants and corresponding
permissive insect host cells from hosts such as Spodoptera
frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes
albopictus (mosquito), Drosophila melanogaster (fruit fly), and
Bombyx mori have been identified. A variety of viral strains for
transfection are publicly available, e.g., the L-1 variant of
Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV,
and such viruses may be used as the virus herein according to the
present invention, particularly for transfection of Spodoptera
frugiperda cells.
Plant cell cultures of cotton, corn, potato, soybean, petunia,
tomato, Arabidopsis and tobacco can also be used as hosts. Cloning
and expression vectors useful in the production of proteins in
plant cell culture are known to those of skill in the art. See e.g.
Hiatt et al., Nature (1989) 342: 76-78, Owen et al. (1992)
Bio/Technology 10: 790-794, Artsaenko et al. (1995) The Plant J 8:
745-750, and Fecker et al. (1996) Plant Mol Biol 32: 979-986.
However, interest has been greatest in vertebrate cells, and
propagation of vertebrate cells in culture (tissue culture) has
become a routine procedure. Examples of useful mammalian host cell
lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC
CRL 1651); human embryonic kidney line (293 or 293 cells subcloned
for growth in suspension culture, Graham et al., J. Gen Virol. 36:
59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese
hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad.
Sci. USA 77: 4216 (1980)); mouse sertoli cells (TM4, Mather, Biol.
Reprod. 23: 243-251 (1980)); monkey kidney cells (CVI ATCC CCL 70);
African green monkey kidney cells (VERO-76, ATCC CRL1587); human
cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells
(MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL
1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep
G2,1413 8065); mouse mammary tumor (MMT 060562, ATCC CCL5 1); TRI
cells (Mather et al., Annals N. Y Acad. Sci. (1982) 383: 44-68);
MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).
In a further embodiment the invention provides a process for the
production of an antibody construct of the invention, said process
comprising culturing a host cell of the invention under conditions
allowing the expression of the antibody construct of the invention
and recovering the produced antibody construct from the
culture.
As used herein, the term "culturing" refers to the in vitro
maintenance, differentiation, growth, proliferation and/or
propagation of cells under suitable conditions in a medium. The
term "expression" includes any step involved in the production of
an antibody construct of the invention including, but not limited
to, transcription, post-transcriptional modification, translation,
post-translational modification, and secretion.
When using recombinant techniques, the antibody construct can be
produced intracellularly, in the periplasmic space, or directly
secreted into the medium. If the antibody construct is produced
intracellularly, as a first step, the particulate debris, either
host cells or lysed fragments, are removed, for example, by
centrifugation or ultrafiltration. Carter et al., Bio/Technology
10: 163-167 (1992) describe a procedure for isolating antibodies
which are secreted to the periplasmic space of E. coli. Briefly,
cell paste is thawed in the presence of sodium acetate (pH 3.5),
EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.
Cell debris can be removed by centrifugation. Where the antibody is
secreted into the medium, supernatants from such expression systems
are generally first concentrated using a commercially available
protein concentration filter, for example, an Amicon or Millipore
Pellicon ultrafiltration unit. A protease inhibitor such as PMSF
may be included in any of the foregoing steps to inhibit
proteolysis and antibiotics may be included to prevent the growth
of adventitious contaminants.
The antibody construct of the invention prepared from the host
cells can be recovered or purified using, for example,
hydroxylapatite chromatography, gel electrophoresis, dialysis, and
affinity chromatography. Other techniques for protein purification
such as fractionation on an ion-exchange column, ethanol
precipitation, Reverse Phase HPLC, chromatography on silica,
chromatography on heparin SEPHAROSE.TM., chromatography on an anion
or cation exchange resin (such as a polyaspartic acid column),
chromato-focusing, SDS-PAGE, and ammonium sulfate precipitation are
also available depending on the antibody to be recovered. Where the
antibody construct of the invention comprises a CH3 domain, the
Bakerbond ABX resin (J. T. Baker, Phillipsburg, N.J.) is useful for
purification.
Affinity chromatography is a preferred purification technique. The
matrix to which the affinity ligand is attached is most often
agarose, but other matrices are available. Mechanically stable
matrices such as controlled pore glass or poly (styrenedivinyl)
benzene allow for faster flow rates and shorter processing times
than can be achieved with agarose.
Moreover, the invention provides a pharmaceutical composition
comprising an antibody construct of the invention or an antibody
construct produced according to the process of the invention.
As used herein, the term "pharmaceutical composition" relates to a
composition which is suitable for administration to a patient,
preferably a human patient. The particularly preferred
pharmaceutical composition of this invention comprises one or a
plurality of the antibody construct(s) of the invention, preferably
in a therapeutically effective amount. Preferably, the
pharmaceutical composition further comprises suitable formulations
of one or more (pharmaceutically effective) carriers, stabilizers,
excipients, diluents, solubilizers, surfactants, emulsifiers,
preservatives and/or adjuvants. Acceptable constituents of the
composition are preferably nontoxic to recipients at the dosages
and concentrations employed. Pharmaceutical compositions of the
invention include, but are not limited to, liquid, frozen, and
lyophilized compositions.
The inventive compositions may comprise a pharmaceutically
acceptable carrier. In general, as used herein, "pharmaceutically
acceptable carrier" means any and all aqueous and non-aqueous
solutions, sterile solutions, solvents, buffers, e.g. phosphate
buffered saline (PBS) solutions, water, suspensions, emulsions,
such as oil/water emulsions, various types of wetting agents,
liposomes, dispersion media and coatings, which are compatible with
pharmaceutical administration, in particular with parenteral
administration. The use of such media and agents in pharmaceutical
compositions is well known in the art, and the compositions
comprising such carriers can be formulated by well-known
conventional methods.
Certain embodiments provide pharmaceutical compositions comprising
the antibody construct of the invention and further one or more
excipients such as those illustratively described in this section
and elsewhere herein. Excipients can be used in the invention in
this regard for a wide variety of purposes, such as adjusting
physical, chemical, or biological properties of formulations, such
as adjustment of viscosity, and or processes of the invention to
improve effectiveness and or to stabilize such formulations and
processes against degradation and spoilage due to, for instance,
stresses that occur during manufacturing, shipping, storage,
pre-use preparation, administration, and thereafter.
In certain embodiments, the pharmaceutical composition may contain
formulation materials for the purpose of modifying, maintaining or
preserving, e.g., the pH, osmolarity, viscosity, clarity, color,
isotonicity, odor, sterility, stability, rate of dissolution or
release, adsorption or penetration of the composition (see,
REMINGTON'S PHARMACEUTICAL SCIENCES, 18" Edition, (A. R. Genrmo,
ed.), 1990, Mack Publishing Company). In such embodiments, suitable
formulation materials may include, but are not limited to:
amino acids such as glycine, alanine, glutamine, asparagine,
threonine, proline, 2-phenylalanine, including charged amino acids,
preferably lysine, lysine acetate, arginine, glutamate and/or
histidine
antimicrobials such as antibacterial and antifungal agents
antioxidants such as ascorbic acid, methionine, sodium sulfite or
sodium hydrogen-sulfite;
buffers, buffer systems and buffering agents which are used to
maintain the composition at physiological pH or at a slightly lower
pH, typically within a pH range of from about 5 to about 8 or 9;
examples of buffers are borate, bicarbonate, Tris-HCl, citrates,
phosphates or other organic acids, succinate, phosphate, histidine
and acetate; for example Tris buffer of about pH 7.0-8.5, or
acetate buffer of about pH 4.0-5.5; non-aqueous solvents such as
propylene glycol, polyethylene glycol, vegetable oils such as olive
oil, and injectable organic esters such as ethyl oleate; aqueous
carriers including water, alcoholic/aqueous solutions, emulsions or
suspensions, including saline and buffered media;
biodegradable polymers such as polyesters;
bulking agents such as mannitol or glycine;
chelating agents such as ethylenediamine tetraacetic acid
(EDTA);
isotonic and absorption delaying agents;
complexing agents such as caffeine, polyvinylpyrrolidone,
beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin) fillers;
monosaccharides; disaccharides; and other carbohydrates (such as
glucose, mannose or dextrins); carbohydrates may be non-reducing
sugars, preferably trehalose, sucrose, octasulfate, sorbitol or
xylitol;
(low molecular weight) proteins, polypeptides or proteinaceous
carriers such as human or bovine serum albumin, gelatin or
immunoglobulins, preferably of human origin; coloring and
flavouring agents;
sulfur containing reducing agents, such as glutathione, thioctic
acid, sodium thioglycolate, thioglycerol, [alpha]-monothioglycerol,
and sodium thio sulfate diluting agents;
emulsifying agents;
hydrophilic polymers such as polyvinylpyrrolidone)
salt-forming counter-ions such as sodium;
preservatives such as antimicrobials, anti-oxidants, chelating
agents, inert gases and the like; examples are: benzalkonium
chloride, benzoic acid, salicylic acid, thimerosal, phenethyl
alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid
or hydrogen peroxide);
metal complexes such as Zn-protein complexes;
solvents and co-solvents (such as glycerin, propylene glycol or
polyethylene glycol); sugars and sugar alcohols, such as trehalose,
sucrose, octasulfate, mannitol, sorbitol or xylitol stachyose,
mannose, sorbose, xylose, ribose, myoinisitose, galactose,
lactitol, ribitol, myoinisitol, galactitol, glycerol, cyclitols
(e.g., inositol), polyethylene glycol; and polyhydric sugar
alcohols;
suspending agents;
surfactants or wetting agents such as pluronics, PEG, sorbitan
esters, polysorbates such as polysorbate 20, polysorbate, triton,
tromethamine, lecithin, cholesterol, tyloxapal; surfactants may be
detergents, preferably with a molecular weight of >1.2 KD and/or
a polyether, preferably with a molecular weight of >3 KD;
non-limiting examples for preferred detergents are Tween 20, Tween
40, Tween 60, Tween 80 and Tween 85; non-limiting examples for
preferred polyethers are PEG 3000, PEG 3350, PEG 4000 and PEG
5000;
stability enhancing agents such as sucrose or sorbitol;
tonicity enhancing agents such as alkali metal halides, preferably
sodium or potassium chloride, mannitol sorbitol;
parenteral delivery vehicles including sodium chloride solution,
Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's,
or fixed oils;
intravenous delivery vehicles including fluid and nutrient
replenishers, electrolyte replenishers (such as those based on
Ringer's dextrose).
It is evident to those skilled in the art that the different
constituents of the pharmaceutical composition (e.g., those listed
above) can have different effects, for example, and amino acid can
act as a buffer, a stabilizer and/or an antioxidant; mannitol can
act as a bulking agent and/or a tonicity enhancing agent; sodium
chloride can act as delivery vehicle and/or tonicity enhancing
agent; etc.
It is envisaged that the composition of the invention might
comprise, in addition to the polypeptide of the invention defined
herein, further biologically active agents, depending on the
intended use of the composition. Such agents might be drugs acting
on the gastro-intestinal system, drugs acting as cytostatica, drugs
preventing hyperurikemia, drugs inhibiting immunoreactions (e.g.
corticosteroids), drugs modulating the inflammatory response, drugs
acting on the circulatory system and/or agents such as cytokines
known in the art. It is also envisaged that the antibody construct
of the present invention is applied in a co-therapy, i.e., in
combination with another anti-cancer medicament.
In certain embodiments, the optimal pharmaceutical composition will
be determined by one skilled in the art depending upon, for
example, the intended route of administration, delivery format and
desired dosage. See, for example, REMINGTON'S PHARMACEUTICAL
SCIENCES, supra. In certain embodiments, such compositions may
influence the physical state, stability, rate of in vivo release
and rate of in vivo clearance of the antibody construct of the
invention. In certain embodiments, the primary vehicle or carrier
in a pharmaceutical composition may be either aqueous or
non-aqueous in nature. For example, a suitable vehicle or carrier
may be water for injection, physiological saline solution or
artificial cerebrospinal fluid, possibly supplemented with other
materials common in compositions for parenteral administration.
Neutral buffered saline or saline mixed with serum albumin are
further exemplary vehicles. In certain embodiments, the antibody
construct of the invention compositions may be prepared for storage
by mixing the selected composition having the desired degree of
purity with optional formulation agents (REMINGTON'S PHARMACEUTICAL
SCIENCES, supra) in the form of a lyophilized cake or an aqueous
solution. Further, in certain embodiments, the antibody construct
of the invention may be formulated as a lyophilizate using
appropriate excipients such as sucrose.
When parenteral administration is contemplated, the therapeutic
compositions for use in this invention may be provided in the form
of a pyrogen-free, parenterally acceptable aqueous solution
comprising the desired antibody construct of the invention in a
pharmaceutically acceptable vehicle. A particularly suitable
vehicle for parenteral injection is sterile distilled water in
which the antibody construct of the invention is formulated as a
sterile, isotonic solution, properly preserved. In certain
embodiments, the preparation can involve the formulation of the
desired molecule with an agent, such as injectable microspheres,
bio-erodible particles, polymeric compounds (such as polylactic
acid or polyglycolic acid), beads or liposomes, that may provide
controlled or sustained release of the product which can be
delivered via depot injection. In certain embodiments, hyaluronic
acid may also be used, having the effect of promoting sustained
duration in the circulation. In certain embodiments, implantable
drug delivery devices may be used to introduce the desired antibody
construct.
Additional pharmaceutical compositions will be evident to those
skilled in the art, including formulations involving the antibody
construct of the invention in sustained- or
controlled-delivery/release formulations. Techniques for
formulating a variety of other sustained- or controlled-delivery
means, such as liposome carriers, bio-erodible microparticles or
porous beads and depot injections, are also known to those skilled
in the art. See, for example, International Patent Application No.
PCT/US93/00829, which describes controlled release of porous
polymeric microparticles for delivery of pharmaceutical
compositions. Sustained-release preparations may include
semipermeable polymer matrices in the form of shaped articles,
e.g., films, or microcapsules. Sustained release matrices may
include polyesters, hydrogels, polylactides (as disclosed in U.S.
Pat. No. 3,773,919 and European Patent Application Publication No.
EP 058481), copolymers of L-glutamic acid and gamma
ethyl-L-glutamate (Sidman et al., 1983, Biopolymers 2:547-556),
poly (2-hydroxyethyl-methacrylate) (Langer et al., 1981, J. Biomed.
Mater. Res. 15:167-277 and Langer, 1982, Chem. Tech. 12:98-105),
ethylene vinyl acetate (Langer et al., 1981, supra) or
poly-D(-)-3-hydroxybutyric acid (European Patent Application
Publication No. EP 133,988). Sustained release compositions may
also include liposomes that can be prepared by any of several
methods known in the art. See, e.g., Eppstein et al., 1985, Proc.
Natl. Acad. Sci. U.S.A. 82:3688-3692; European Patent Application
Publication Nos. EP 036,676; EP 088,046 and EP 143,949.
The antibody construct may also be entrapped in microcapsules
prepared, for example, by coacervation techniques or by interfacial
polymerization (for example, hydroxymethylcellulose or
gelatine-microcapsules and poly (methylmethacylate) microcapsules,
respectively), in colloidal drug delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nanoparticles and
nanocapsules), or in macroemulsions. Such techniques are disclosed
in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A.,
Ed., (1980).
Pharmaceutical compositions used for in vivo administration are
typically provided as sterile preparations. Sterilization can be
accomplished by filtration through sterile filtration membranes.
When the composition is lyophilized, sterilization using this
method may be conducted either prior to or following lyophilization
and reconstitution. Compositions for parenteral administration can
be stored in lyophilized form or in a solution. Parenteral
compositions generally are placed into a container having a sterile
access port, for example, an intravenous solution bag or vial
having a stopper pierceable by a hypodermic injection needle.
Another aspect of the invention includes self-buffering antibody
construct of the invention formulations, which can be used as
pharmaceutical compositions, as described in international patent
application WO 06138181 A2 (PCT/US2006/022599). A variety of
expositions are available on protein stabilization and formulation
materials and methods useful in this regard, such as Arakawa et
al., "Solvent interactions in pharmaceutical formulations," Pharm
Res. 8(3): 285-91 (1991); Kendrick et al., "Physical stabilization
of proteins in aqueous solution" in: RATIONAL DESIGN OF STABLE
PROTEIN FORMULATIONS: THEORY AND PRACTICE, Carpenter and Manning,
eds. Pharmaceutical Biotechnology. 13: 61-84 (2002), and Randolph
et al., "Surfactant-protein interactions", Pharm Biotechnol. 13:
159-75 (2002), see particularly the parts pertinent to excipients
and processes of the same for self-buffering protein formulations
in accordance with the current invention, especially as to protein
pharmaceutical products and processes for veterinary and/or human
medical uses.
Salts may be used in accordance with certain embodiments of the
invention to, for example, adjust the ionic strength and/or the
isotonicity of a formulation and/or to improve the solubility
and/or physical stability of a protein or other ingredient of a
composition in accordance with the invention. As is well known,
ions can stabilize the native state of proteins by binding to
charged residues on the protein's surface and by shielding charged
and polar groups in the protein and reducing the strength of their
electrostatic interactions, attractive, and repulsive interactions.
Ions also can stabilize the denatured state of a protein by binding
to, in particular, the denatured peptide linkages (--CONH) of the
protein. Furthermore, ionic interaction with charged and polar
groups in a protein also can reduce intermolecular electrostatic
interactions and, thereby, prevent or reduce protein aggregation
and insolubility.
Ionic species differ significantly in their effects on proteins. A
number of categorical rankings of ions and their effects on
proteins have been developed that can be used in formulating
pharmaceutical compositions in accordance with the invention. One
example is the Hofmeister series, which ranks ionic and polar
non-ionic solutes by their effect on the conformational stability
of proteins in solution. Stabilizing solutes are referred to as
"kosmotropic". Destabilizing solutes are referred to as
"chaotropic". Kosmotropes commonly are used at high concentrations
(e.g., .gtoreq.1 molar ammonium sulfate) to precipitate proteins
from solution ("salting-out"). Chaotropes commonly are used to
denture and/or to solubilize proteins ("salting-in"). The relative
effectiveness of ions to "salt-in" and "salt-out" defines their
position in the Hofmeister series.
Free amino acids can be used in the antibody construct of the
invention formulations in accordance with various embodiments of
the invention as bulking agents, stabilizers, and antioxidants, as
well as other standard uses. Lysine, proline, serine, and alanine
can be used for stabilizing proteins in a formulation. Glycine is
useful in lyophilization to ensure correct cake structure and
properties. Arginine may be useful to inhibit protein aggregation,
in both liquid and lyophilized formulations. Methionine is useful
as an antioxidant.
Polyols include sugars, e.g., mannitol, sucrose, and sorbitol and
polyhydric alcohols such as, for instance, glycerol and propylene
glycol, and, for purposes of discussion herein, polyethylene glycol
(PEG) and related substances. Polyols are kosmotropic. They are
useful stabilizing agents in both liquid and lyophilized
formulations to protect proteins from physical and chemical
degradation processes. Polyols also are useful for adjusting the
tonicity of formulations. Among polyols useful in select
embodiments of the invention is mannitol, commonly used to ensure
structural stability of the cake in lyophilized formulations. It
ensures structural stability to the cake. It is generally used with
a lyoprotectant, e.g., sucrose. Sorbitol and sucrose are among
preferred agents for adjusting tonicity and as stabilizers to
protect against freeze-thaw stresses during transport or the
preparation of bulks during the manufacturing process. Reducing
sugars (which contain free aldehyde or ketone groups), such as
glucose and lactose, can glycate surface lysine and arginine
residues. Therefore, they generally are not among preferred polyols
for use in accordance with the invention. In addition, sugars that
form such reactive species, such as sucrose, which is hydrolyzed to
fructose and glucose under acidic conditions, and consequently
engenders glycation, also is not among preferred polyols of the
invention in this regard. PEG is useful to stabilize proteins and
as a cryoprotectant and can be used in the invention in this
regard.
Embodiments of the antibody construct of the invention formulations
further comprise surfactants. Protein molecules may be susceptible
to adsorption on surfaces and to denaturation and consequent
aggregation at air-liquid, solid-liquid, and liquid-liquid
interfaces. These effects generally scale inversely with protein
concentration. These deleterious interactions generally scale
inversely with protein concentration and typically are exacerbated
by physical agitation, such as that generated during the shipping
and handling of a product. Surfactants routinely are used to
prevent, minimize, or reduce surface adsorption. Useful surfactants
in the invention in this regard include polysorbate 20, polysorbate
80, other fatty acid esters of sorbitan polyethoxylates, and
poloxamer 188. Surfactants also are commonly used to control
protein conformational stability. The use of surfactants in this
regard is protein-specific since, any given surfactant typically
will stabilize some proteins and destabilize others.
Polysorbates are susceptible to oxidative degradation and often, as
supplied, contain sufficient quantities of peroxides to cause
oxidation of protein residue side-chains, especially methionine.
Consequently, polysorbates should be used carefully, and when used,
should be employed at their lowest effective concentration. In this
regard, polysorbates exemplify the general rule that excipients
should be used in their lowest effective concentrations.
Embodiments of the antibody construct of the invention formulations
further comprise one or more antioxidants. To some extent
deleterious oxidation of proteins can be prevented in
pharmaceutical formulations by maintaining proper levels of ambient
oxygen and temperature and by avoiding exposure to light.
Antioxidant excipients can be used as well to prevent oxidative
degradation of proteins. Among useful antioxidants in this regard
are reducing agents, oxygen/free-radical scavengers, and chelating
agents. Antioxidants for use in therapeutic protein formulations in
accordance with the invention preferably are water-soluble and
maintain their activity throughout the shelf life of a product.
EDTA is a preferred antioxidant in accordance with the invention in
this regard. Antioxidants can damage proteins. For instance,
reducing agents, such as glutathione in particular, can disrupt
intramolecular disulfide linkages. Thus, antioxidants for use in
the invention are selected to, among other things, eliminate or
sufficiently reduce the possibility of themselves damaging proteins
in the formulation.
Formulations in accordance with the invention may include metal
ions that are protein co-factors and that are necessary to form
protein coordination complexes, such as zinc necessary to form
certain insulin suspensions. Metal ions also can inhibit some
processes that degrade proteins. However, metal ions also catalyze
physical and chemical processes that degrade proteins. Magnesium
ions (10-120 mM) can be used to inhibit isomerization of aspartic
acid to isoaspartic acid. Ca.sup.+2 ions (up to 100 mM) can
increase the stability of human deoxyribonuclease. Mg.sup.+2,
Mn.sup.+2, and Zn.sup.+2, however, can destabilize rhDNase.
Similarly, Ca.sup.+2 and Sr.sup.+2 can stabilize Factor VIII, it
can be destabilized by Mg.sup.+2, Mn.sup.+2 and Zn.sup.+2,
Cu.sup.+2 and Fe.sup.+2, and its aggregation can be increased by
Al.sup.+3 ions.
Embodiments of the antibody construct of the invention formulations
further comprise one or more preservatives. Preservatives are
necessary when developing multi-dose parenteral formulations that
involve more than one extraction from the same container. Their
primary function is to inhibit microbial growth and ensure product
sterility throughout the shelf-life or term of use of the drug
product. Commonly used preservatives include benzyl alcohol, phenol
and m-cresol. Although preservatives have a long history of use
with small-molecule parenterals, the development of protein
formulations that includes preservatives can be challenging.
Preservatives almost always have a destabilizing effect
(aggregation) on proteins, and this has become a major factor in
limiting their use in multi-dose protein formulations. To date,
most protein drugs have been formulated for single-use only.
However, when multi-dose formulations are possible, they have the
added advantage of enabling patient convenience, and increased
marketability. A good example is that of human growth hormone (hGH)
where the development of preserved formulations has led to
commercialization of more convenient, multi-use injection pen
presentations. At least four such pen devices containing preserved
formulations of hGH are currently available on the market.
Norditropin (liquid, Novo Nordisk), Nutropin AQ (liquid, Genentech)
& Genotropin (lyophilized--dual chamber cartridge, Pharmacia
& Upjohn) contain phenol while Somatrope (Eli Lilly) is
formulated with m-cresol. Several aspects need to be considered
during the formulation and development of preserved dosage forms.
The effective preservative concentration in the drug product must
be optimized. This requires testing a given preservative in the
dosage form with concentration ranges that confer anti-microbial
effectiveness without compromising protein stability.
As might be expected, development of liquid formulations containing
preservatives are more challenging than lyophilized formulations.
Freeze-dried products can be lyophilized without the preservative
and reconstituted with a preservative containing diluent at the
time of use. This shortens the time for which a preservative is in
contact with the protein, significantly minimizing the associated
stability risks. With liquid formulations, preservative
effectiveness and stability should be maintained over the entire
product shelf-life (about 18 to 24 months). An important point to
note is that preservative effectiveness should be demonstrated in
the final formulation containing the active drug and all excipient
components.
The antibody constructs disclosed herein may also be formulated as
immuno-liposomes. A "liposome" is a small vesicle composed of
various types of lipids, phospholipids and/or surfactant which is
useful for delivery of a drug to a mammal. The components of the
liposome are commonly arranged in a bilayer formation, similar to
the lipid arrangement of biological membranes. Liposomes containing
the antibody construct are prepared by methods known in the art,
such as described in Epstein et al., Proc. Natl. Acad. Sci. USA,
82: 3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA, 77: 4030
(1980); U.S. Pat. Nos. 4,485,045 and 4,544,545; and WO 97/38731.
Liposomes with enhanced circulation time are disclosed in U.S. Pat.
No. 5,013,556. Particularly useful liposomes can be generated by
the reverse phase evaporation method with a lipid composition
comprising phosphatidylcholine, cholesterol and PEG-derivatized
phosphatidylethanolamine (PEG-PE). Liposomes are extruded through
filters of defined pore size to yield liposomes with the desired
diameter. Fab' fragments of the antibody construct of the present
invention can be conjugated to the liposomes as described in Martin
et al. J. Biol. Chem. 257: 286-288 (1982) via a disulfide
interchange reaction. A chemotherapeutic agent is optionally
contained within the liposome. See Gabizon et al. J. National
Cancer Inst. 81 (19) 1484 (1989).
Once the pharmaceutical composition has been formulated, it may be
stored in sterile vials as a solution, suspension, gel, emulsion,
solid, crystal, or as a dehydrated or lyophilized powder. Such
formulations may be stored either in a ready-to-use form or in a
form (e.g., lyophilized) that is reconstituted prior to
administration.
The biological activity of the pharmaceutical composition defined
herein can be determined for instance by cytotoxicity assays, as
described in the following examples, in WO 99/54440 or by Schlereth
et al. (Cancer Immunol. Immunother. 20 (2005), 1-12). "Efficacy" or
"in vivo efficacy" as used herein refers to the response to therapy
by the pharmaceutical composition of the invention, using e.g.
standardized NCI response criteria. The success or in vivo efficacy
of the therapy using a pharmaceutical composition of the invention
refers to the effectiveness of the composition for its intended
purpose, i.e. the ability of the composition to cause its desired
effect, i.e. depletion of pathologic cells, e.g. tumor cells. The
in vivo efficacy may be monitored by established standard methods
for the respective disease entities including, but not limited to
white blood cell counts, differentials, Fluorescence Activated Cell
Sorting, bone marrow aspiration. In addition, various disease
specific clinical chemistry parameters and other established
standard methods may be used. Furthermore, computer-aided
tomography, X-ray, nuclear magnetic resonance tomography (e.g. for
National Cancer Institute-criteria based response assessment
[Cheson B D, Horning S J, Coiffier B, Shipp M A, Fisher R I,
Connors J M, Lister T A, Vose J, Grillo-Lopez A, Hagenbeek A,
Cabanillas F, Klippensten D, Hiddemann W, Castellino R, Harris N L,
Armitage J O, Carter W, Hoppe R, Canellos G P. Report of an
international workshop to standardize response criteria for
non-Hodgkin's lymphomas. NCI Sponsored International Working Group.
J Clin Oncol. 1999 April; 17(4):1244]), positron-emission
tomography scanning, white blood cell counts, differentials,
Fluorescence Activated Cell Sorting, bone marrow aspiration, lymph
node biopsies/histologies, and various lymphoma specific clinical
chemistry parameters (e.g. lactate dehydrogenase) and other
established standard methods may be used.
Another major challenge in the development of drugs such as the
pharmaceutical composition of the invention is the predictable
modulation of pharmacokinetic properties. To this end, a
pharmacokinetic profile of the drug candidate, i.e. a profile of
the pharmacokinetic parameters that affect the ability of a
particular drug to treat a given condition, can be established.
Pharmacokinetic parameters of the drug influencing the ability of a
drug for treating a certain disease entity include, but are not
limited to: half-life, volume of distribution, hepatic first-pass
metabolism and the degree of blood serum binding. The efficacy of a
given drug agent can be influenced by each of the parameters
mentioned above.
"Half-life" means the time where 50% of an administered drug are
eliminated through biological processes, e.g. metabolism,
excretion, etc. By "hepatic first-pass metabolism" is meant the
propensity of a drug to be metabolized upon first contact with the
liver, i.e. during its first pass through the liver. "Volume of
distribution" means the degree of retention of a drug throughout
the various compartments of the body, like e.g. intracellular and
extracellular spaces, tissues and organs, etc. and the distribution
of the drug within these compartments. "Degree of blood serum
binding" means the propensity of a drug to interact with and bind
to blood serum proteins, such as albumin, leading to a reduction or
loss of biological activity of the drug.
Pharmacokinetic parameters also include bioavailability, lag time
(TIag), Tmax, absorption rates, more onset and/or Cmax for a given
amount of drug administered. "Bioavailability" means the amount of
a drug in the blood compartment. "Lag time" means the time delay
between the administration of the drug and its detection and
measurability in blood or plasma. "Tmax" is the time after which
maximal blood concentration of the drug is reached, and "Cmax" is
the blood concentration maximally obtained with a given drug. The
time to reach a blood or tissue concentration of the drug which is
required for its biological effect is influenced by all parameters.
Pharmacokinetic parameters of bispecific antibody constructs
exhibiting cross-species specificity, which may be determined in
preclinical animal testing in non-chimpanzee primates as outlined
above, are also set forth e.g. in the publication by Schlereth et
al. (Cancer Immunol. Immunother. 20 (2005), 1-12).
One embodiment provides the antibody construct of the invention or
the antibody construct produced according to the process of the
invention for use in the prevention, treatment or amelioration of a
tumor or cancer disease or of a metastatic cancer disease.
The formulations described herein are useful as pharmaceutical
compositions in the treatment, amelioration and/or prevention of
the pathological medical condition as described herein in a patient
in need thereof. The term "treatment" refers to both therapeutic
treatment and prophylactic or preventative measures. Treatment
includes the application or administration of the formulation to
the body, an isolated tissue, or cell from a patient who has a
disease/disorder, a symptom of a disease/disorder, or a
predisposition toward a disease/disorder, with the purpose to cure,
heal, alleviate, relieve, alter, remedy, ameliorate, improve, or
affect the disease, the symptom of the disease, or the
predisposition toward the disease.
The term "amelioration" as used herein refers to any improvement of
the disease state of a patient having a tumor or cancer or a
metastatic cancer as specified herein below, by the administration
of an antibody construct according to the invention to a subject in
need thereof. Such an improvement may also be seen as a slowing or
stopping of the p progression of the tumor or cancer or metastatic
cancer of the patient. The term "prevention" as used herein means
the avoidance of the occurrence or re-occurrence of a patient
having a tumor or cancer or a metastatic cancer as specified herein
below, by the administration of an antibody construct according to
the invention to a subject in need thereof.
The term "disease" refers to any condition that would benefit from
treatment with the antibody construct or the pharmaceutic
composition described herein. This includes chronic and acute
disorders or diseases including those pathological conditions that
predispose the mammal to the disease in question.
A "neoplasm" is is an abnormal growth of tissue, usually but not
always forming a mass. When also forming a mass, it is commonly
referred to as a "tumor". Neoplasms or tumors can be benign,
potentially malignant (pre-cancerous), or malignant. Malignant
neoplasms are commonly called cancer. They usually invade and
destroy the surrounding tissue and may form metastases, i.e., they
spread to other parts, tissues or organs of the body. Hence, the
term "metatstatic cancer" encompasses metastases to other tissues
or organs than the one of the original tumor. Lymphomas and
leukemias are lymphoid neoplasms. For the purposes of the present
invention, they are also encompassed by the terms "tumor" or
"cancer".
In a preferred embodiment of the invention, the tumor or cancer
disease is selected from the group including, but not limited to,
(or consisting of) lung cancer, preferably SCLC, breast, cervical,
colon, colorectal, endometrial, head and neck, liver, ovarian,
pancreatic, prostate, skin, gastric, testis, thyroid, adrenal,
renal, bladder, uterine, esophageal, urothelial and brain tumor or
cancer, lymphoma, carcinoma, and sarcoma, and a metastatic cancer
disease derived from any of the foregoing.
More specifically, the tumor or cancer disease can be selected from
the group consisting of small cell lung cancer (SCLC), non-small
ceil lung cancer (NSCLC), glioma, glioblastoma, melanoma,
neuroendocrine prostate cancer, neuroendocrine pancreatic cancer,
hepatoblastoma, and hepatocellular carcinoma. The metastatic cancer
disease can be derived from any of the foregoing.
The invention also provides a method for the treatment or
amelioration of tumor or cancer disease or a metastatic cancer
disease, comprising the step of administering to a subject in need
thereof the antibody construct of the invention or the antibody
construct produced according to the process of the invention.
The terms "subject in need" or those "in need of treatment"
includes those already with the disorder, as well as those in which
the disorder is to be prevented. The subject in need or "patient"
includes human and other mammalian subjects that receive either
prophylactic or therapeutic treatment.
The antibody construct of the invention will generally be designed
for specific routes and methods of administration, for specific
dosages and frequencies of administration, for specific treatments
of specific diseases, with ranges of bio-availability and
persistence, among other things. The materials of the composition
are preferably formulated in concentrations that are acceptable for
the site of administration.
Formulations and compositions thus may be designed in accordance
with the invention for delivery by any suitable route of
administration. In the context of the present invention, the routes
of administration include, but are not limited to
topical routes (such as epicutaneous, inhalational, nasal,
opthalmic, auricular/aural, vaginal, mucosal);
enteral routes (such as oral, gastrointestinal, sublingual,
sublabial, buccal, rectal); and
parenteral routes (such as intravenous, intraarterial,
intraosseous, intramuscular, intracerebral,
intracerebroventricular, epidural, intrathecal, subcutaneous,
intraperitoneal, extra-amniotic, intraarticular, intracardiac,
intradermal, intralesional, intrauterine, intravesical,
intravitreal, transdermal, intranasal, transmucosal, intrasynovial,
intraluminal).
The pharmaceutical compositions and the antibody construct of this
invention are particularly useful for parenteral administration,
e.g., subcutaneous or intravenous delivery, for example by
injection such as bolus injection, or by infusion such as
continuous infusion. Pharmaceutical compositions may be
administered using a medical device. Examples of medical devices
for administering pharmaceutical compositions are described in U.S.
Pat. Nos. 4,475,196; 4,439,196; 4,447,224; 4,447, 233; 4,486,194;
4,487,603; 4,596,556; 4,790,824; 4,941,880; 5,064,413; 5,312,335;
5,312,335; 5,383,851; and 5,399,163.
In particular, the present invention provides for an uninterrupted
administration of the suitable composition. As a non-limiting
example, uninterrupted or substantially uninterrupted, i.e.
continuous administration may be realized by a small pump system
worn by the patient for metering the influx of therapeutic agent
into the body of the patient. The pharmaceutical composition
comprising the antibody construct of the invention can be
administered by using said pump systems. Such pump systems are
generally known in the art, and commonly rely on periodic exchange
of cartridges containing the therapeutic agent to be infused. When
exchanging the cartridge in such a pump system, a temporary
interruption of the otherwise uninterrupted flow of therapeutic
agent into the body of the patient may ensue. In such a case, the
phase of administration prior to cartridge replacement and the
phase of administration following cartridge replacement would still
be considered within the meaning of the pharmaceutical means and
methods of the invention together make up one "uninterrupted
administration" of such therapeutic agent.
The continuous or uninterrupted administration of the antibody
constructs of the invention may be intravenous or subcutaneous by
way of a fluid delivery device or small pump system including a
fluid driving mechanism for driving fluid out of a reservoir and an
actuating mechanism for actuating the driving mechanism. Pump
systems for subcutaneous administration may include a needle or a
cannula for penetrating the skin of a patient and delivering the
suitable composition into the patient's body. Said pump systems may
be directly fixed or attached to the skin of the patient
independently of a vein, artery or blood vessel, thereby allowing a
direct contact between the pump system and the skin of the patient.
The pump system can be attached to the skin of the patient for 24
hours up to several days. The pump system may be of small size with
a reservoir for small volumes. As a non-limiting example, the
volume of the reservoir for the suitable pharmaceutical composition
to be administered can be between 0.1 and 50 ml.
The continuous administration may also be transdermal by way of a
patch worn on the skin and replaced at intervals. One of skill in
the art is aware of patch systems for drug delivery suitable for
this purpose. It is of note that transdermal administration is
especially amenable to uninterrupted administration, as exchange of
a first exhausted patch can advantageously be accomplished
simultaneously with the placement of a new, second patch, for
example on the surface of the skin immediately adjacent to the
first exhausted patch and immediately prior to removal of the first
exhausted patch. Issues of flow interruption or power cell failure
do not arise.
If the pharmaceutical composition has been lyophilized, the
lyophilized material is first reconstituted in an appropriate
liquid prior to administration. The lyophilized material may be
reconstituted in, e.g., bacteriostatic water for injection (BWFI),
physiological saline, phosphate buffered saline (PBS), or the same
formulation the protein had been in prior to lyophilization.
The compositions of the present invention can be administered to
the subject at a suitable dose which can be determined e.g. by dose
escalating studies by administration of increasing doses of the
antibody construct of the invention exhibiting cross-species
specificity described herein to non-chimpanzee primates, for
instance macaques. As set forth above, the antibody construct of
the invention exhibiting cross-species specificity described herein
can be advantageously used in identical form in preclinical testing
in non-chimpanzee primates and as drug in humans. The dosage
regimen will be determined by the attending physician and clinical
factors. As is well known in the medical arts, dosages for any one
patient depend upon many factors, including the patient's size,
body surface area, age, the particular compound to be administered,
sex, time and route of administration, general health, and other
drugs being administered concurrently.
The term "effective dose" or "effective dosage" is defined as an
amount sufficient to achieve or at least partially achieve the
desired effect. The term "therapeutically effective dose" is
defined as an amount sufficient to cure or at least partially
arrest the disease and its complications in a patient already
suffering from the disease. Amounts or doses effective for this use
will depend on the condition to be treated (the indication), the
delivered antibody construct, the therapeutic context and
objectives, the severity of the disease, prior therapy, the
patient's clinical history and response to the therapeutic agent,
the route of administration, the size (body weight, body surface or
organ size) and/or condition (the age and general health) of the
patient, and the general state of the patient's own immune system.
The proper dose can be adjusted according to the judgment of the
attending physician such that it can be administered to the patient
once or over a series of administrations, and in order to obtain
the optimal therapeutic effect.
A typical dosage may range from about 0.1 .mu.g/kg to up to about
30 mg/kg or more, depending on the factors mentioned above. In
specific embodiments, the dosage may range from 1.0 .mu.g/kg up to
about 20 mg/kg, optionally from 10 .mu.g/kg up to about 10 mg/kg or
from 100 .mu.g/kg up to about 5 mg/kg.
A therapeutic effective amount of an antibody construct of the
invention preferably results in a decrease in severity of disease
symptoms, an increase in frequency or duration of disease
symptom-free periods or a prevention of impairment or disability
due to the disease affliction. For treating DLL3-expressing tumors,
a therapeutically effective amount of the antibody construct of the
invention, e.g. an anti-DLL3/anti-CD3 antibody construct,
preferably inhibits cell growth or tumor growth by at least about
20%, at least about 40%, at least about 50%, at least about 60%, at
least about 70%, at least about 80%, or at least about 90% relative
to untreated patients. The ability of a compound to inhibit tumor
growth may be evaluated in an animal model predictive of efficacy
in human tumors.
The pharmaceutical composition can be administered as a sole
therapeutic or in combination with additional therapies such as
anti-cancer therapies as needed, e.g. other proteinaceous and
non-proteinaceous drugs. These drugs may be administered
simultaneously with the composition comprising the antibody
construct of the invention as defined herein or separately before
or after administration of said antibody construct in timely
defined intervals and doses.
The term "effective and non-toxic dose" as used herein refers to a
tolerable dose of an inventive antibody construct which is high
enough to cause depletion of pathologic cells, tumor elimination,
tumor shrinkage or stabilization of disease without or essentially
without major toxic effects. Such effective and non-toxic doses may
be determined e.g. by dose escalation studies described in the art
and should be below the dose inducing severe adverse side events
(dose limiting toxicity, DLT).
The term "toxicity" as used herein refers to the toxic effects of a
drug manifested in adverse events or severe adverse events. These
side events might refer to a lack of tolerability of the drug in
general and/or a lack of local tolerance after administration.
Toxicity could also include teratogenic or carcinogenic effects
caused by the drug.
The term "safety", "in vivo safety" or "tolerability" as used
herein defines the administration of a drug without inducing severe
adverse events directly after administration (local tolerance) and
during a longer period of application of the drug. "Safety", "in
vivo safety" or "tolerability" can be evaluated e.g. at regular
intervals during the treatment and follow-up period. Measurements
include clinical evaluation, e.g. organ manifestations, and
screening of laboratory abnormalities. Clinical evaluation may be
carried out and deviations to normal findings recorded/coded
according to NCI-CTC and/or MedDRA standards. Organ manifestations
may include criteria such as allergy/immunology, blood/bone marrow,
cardiac arrhythmia, coagulation and the like, as set forth e.g. in
the Common Terminology Criteria for adverse events v3.0 (CTCAE).
Laboratory parameters which may be tested include for instance
hematology, clinical chemistry, coagulation profile and urine
analysis and examination of other body fluids such as serum,
plasma, lymphoid or spinal fluid, liquor and the like. Safety can
thus be assessed e.g. by physical examination, imaging techniques
(i.e. ultrasound, x-ray, CT scans, Magnetic Resonance Imaging
(MRI), other measures with technical devices (i.e.
electrocardiogram), vital signs, by measuring laboratory parameters
and recording adverse events. For example, adverse events in
non-chimpanzee primates in the uses and methods according to the
invention may be examined by histopathological and/or histochemical
methods.
The above terms are also referred to e.g. in the Preclinical safety
evaluation of biotechnology-derived pharmaceuticals S6; ICH
Harmonised Tripartite Guideline; ICH Steering Committee meeting on
Jul. 16, 1997.
In a further embodiment, the invention provides a kit comprising an
antibody construct of the invention, an antibody construct produced
according to the process of the invention, a polypeptide of the
invention, a vector of the invention, and/or a host cell of the
invention.
In the context of the present invention, the term "kit" means two
or more components--one of which corresponding to the antibody
construct, the pharmaceutical composition, the vector or the host
cell of the invention--packaged together in a container, recipient
or otherwise. A kit can hence be described as a set of products
and/or utensils that are sufficient to achieve a certain goal,
which can be marketed as a single unit.
The kit may comprise one or more recipients (such as vials,
ampoules, containers, syringes, bottles, bags) of any appropriate
shape, size and material (preferably waterproof, e.g. plastic or
glass) containing the antibody construct or the pharmaceutical
composition of the present invention in an appropriate dosage for
administration (see above). The kit may additionally contain
directions for use (e.g. in the form of a leaflet or instruction
manual), means for administering the antibody construct of the
present invention such as a syringe, pump, infuser or the like,
means for reconstituting the antibody construct of the invention
and/or means for diluting the antibody construct of the
invention.
The invention also provides kits for a single-dose administration
unit. The kit of the invention may also contain a first recipient
comprising a dried/lyophilized antibody construct and a second
recipient comprising an aqueous formulation. In certain embodiments
of this invention, kits containing single-chambered and
multi-chambered pre-filled syringes (e.g., liquid syringes and
lyosyringes) are provided.
The Figures Show:
FIG. 1
Schematic representation of the DLL3 ECD/truncated DLL3 constructs
expressed on CHO cells for epitope mapping. The transmembrane and
the intracellular domain are derived from EpCAM. See Example 1.
FIGS. 2A and 2B
Epitope mapping of the anti-DLL3 antibody constructs. Examples of
bispecific antibody constructs recognizing the EGF-3 domain and the
EGF-4 domain, respectively, as detected by epitope mapping. The
x-axis depicts PE-A (PE=phycoerythrin, A=signal area), and the
y-axis depicts counts. See Example 2.
FIGS. 3A-3C
Cross-Reactivity of anti-DLL3 antibody constructs as detected by
flow cytometry: binding to human and macaque DLL3 and CD3. The
x-axis depicts FL2-H, and the y-axis depicts counts. See Example
5.
FIG. 4
Analysis of anti-DLL3 antibody constructs by flow cytometry:
non-binding to human paralogues DLL1 and DLL4. The x-axis depicts
FL1-H, and the y-axis depicts counts. See Example 6.
FIG. 5
Stability of anti-DLL3 antibody constructs after incubation for 96
hours in human plasma. See Example 11.
FIG. 6
Potency gap between the monomeric and the dimeric isoform of the
anti-DLL3 antibody constructs. See Example 15.
FIG. 7
In vitro internalization assay, carried out as described in Example
16. Antibody construct was pre-incubated on target cells (SHP-77)
in the absence of T cells in order to measure loss of antibody
construct due to internalization. Results suggest that no
significant internalization occurs. The results achieved with a
control target which is well known for its internalization are
shown for comparison at the right.
FIGS. 8A and 8B
Results of the mouse xenograft efficacy study of the SHP-77 model
(FIG. 8A) and the WM266-4 model (FIG. 8B). The tumor volume is
plotted against the time. See Example 18.
FIGS. 9A and 9B
Pharmacokinetics of different HLE antibody constructs (albumin
fusion in FIG. 9A and Fc fusions in FIG. 9B) as determined in
Example 20.
EXAMPLES
The following examples illustrate the invention. These examples
should not be construed as to limit the scope of this invention.
The present invention is limited only by the claims.
Example 1
Generation of CHO cells expressing wild type and truncated DLL3
The extracellular domain of the DLL3 antigen can be subdivided into
different sub-domains or regions that are defined, for the purposes
of Examples 1 and 2, by the following amino acid positions:
Signal peptide: 1-26
N-terminus: 27-175
DSL: 176-215
EGF-1: 216-249
EGF-2: 274-310
EGF-3: 312-351
EGF-4: 353-389
EGF-5: 391-427
EGF-6: 429-465
For the construction of the truncated DLL3 molecules used for
epitope mapping, the sequences of the respective eight
extracellular domains (Signal peptide plus N-terminus, DSL, EGF1,
EGF2, EGF3, EGF4, EGF5 and EGF6) of human DLL3 were stepwise
deleted from the antigen, starting from the N-terminus. The
following molecules were generated; see also FIG. 1: Hu DLL3
ECD/complete ECD SEQ ID NO: 263 Hu DLL3 ECD/up to DSL SEQ ID NO:
264 Hu DLL3 ECD/up to EGF-1 SEQ ID NO: 265 Hu DLL3 ECD/up to EGF-2
SEQ ID NO: 266 Hu DLL3 ECD/up to EGF-3 SEQ ID NO: 267 Hu DLL3
ECD/up to EGF-4 SEQ ID NO: 268 Hu DLL3 ECD/up to EGF-5 SEQ ID NO:
269 Hu DLL3 ECD/only EGF-6 SEQ ID NO: 270
For the generation of CHO cells expressing human, cynomolgus
macaque ("cyno") and truncated human N-terminal V5 tagged DLL3, the
respective coding sequences for human DLL3-ECD (SEQ ID NO: 253; see
also GeneBank accession number NM_016941), cyno DLL3-ECD (SEQ ID
NO: 272) and the seven truncated human N-terminal V5 tagged
DLL3-ECD versions (see above) were cloned into a plasmid designated
pEF-DHFR (pEF-DHFR is described in Raum et al. Cancer Immunol
Immunother 50 (2001) 141-150). For cell surface expression of human
and cyno DLL3 the original signal peptide was used, and for cell
surface expression of the truncated human N-terminal DLL3 a mouse
IgG heavy chain signal peptide was used, followed by a V5 tag. All
mentioned DLL3-ECD sequences were followed in frame by the coding
sequence of an artificial Ser/Gly-linker followed by a domain
derived from the transmembrane/intracellular domain of human EpCAM
(amino acids 266-314 of the sequence as published in GenBank
accession number NM_002354). All cloning procedures were carried
out according to standard protocols (Sambrook, Molecular Cloning; A
Laboratory Manual, 3rd edition, Cold Spring Harbour Laboratory
Press, Cold Spring Harbour, N.Y. (2001)). For each construct, a
corresponding plasmid was transfected into DHFR deficient CHO cells
for eukaryotic expression, as described by Kaufman R. J. (1990)
Methods Enzymol. 185, 537-566.
The expression of human and cyno DLL3 on CHO cells was verified in
a FACS assay using a monoclonal mouse IgG2b anti-human DLL3
antibody. The expression of the seven truncated versions of human
DLL3-ECD was verified using a monoclonal mouse IgG2a anti-v5 tag
antibody. Bound monoclonal antibody was detected with an anti-mouse
IgG Fc-gamma-PE. As negative control, cells were incubated with
isotype control antibody instead of the first antibody. The samples
were measured by flow cytometry.
Example 2
Epitope Mapping of Anti-DLL3 Antibody Constructs
Cells transfected with human DLL3 and with the truncated human DLL3
molecules (see Example 1) were stained with crude, undiluted
periplasmic extract containing bispecific DLL3.times.CD3 antibody
constructs (with the CD3 binding domain being denominated I2C)
fused to a human albumin (variant 1), in PBS/1.5% FCS. Bound
molecules were detected with an in-house mouse monoclonal anti-CD3
binding domain antibody (50 .mu.l) followed by an anti-mouse IgG
Fc-gamma-PE (1:100, 50 .mu.l; Jackson Immunoresearch #115-116-071)
All antibodies were diluted in PBS/1.5% FCS. As negative control,
cells were incubated with PBS/2% FCS instead of the periplasmic
extract. The samples were measured by flow cytometry.
The regions that were recognized by the respective DLL3 binding
domains are indicated in the sequence table (Table 18). Binders
were mapped that were specific for an epitope located within the
N-terminus of DLL3, within the DSL domain, and within the different
EGF domains.
FIGS. 2A and 2B shows two exemplary binders which bind to a DLL3
epitope comprised within the EGF-3 region (loss of the FACS signal
in the respective truncated DLL3 constructs not comprising EGF-3).
FIGS. 2A and 2B also shows an exemplary binder which binds to a
DLL3 epitope comprised within the EGF-3 region (loss of the FACS
signal in the respective truncated DLL3 constructs not comprising
EGF-4).
Some of the binders are described to be specific for an epitope
located with the region denominated EGF-5/[6]. The squared brackets
are meant to indicate that the FACS signal of the binder is
decreased (i.e., neither fully present nor fully lost) for the
truncated DLL3 construct having only the EGF-6 domain left (last
construct in FIG. 1).
The bispecific DLL3.times.CD3 constructs used in the following
examples are chosen from those constructs having "I2C" as the CD3
binding domain and having a C-terminal fusion to a wild-type human
serum albumin, see e.g. SEQ ID NOs: 224-230, 233-235, 238-241.
Example 3
Biacore-Based Determination of Antibody Affinity to Human and
Cynomolgus DLL3
Biacore analysis experiments were performed using recombinant
human/cyno DLL3-ECD fusion proteins with chicken albumin to
determine target binding of the antibody constructs of the
invention.
In detail, CM5 Sensor Chips (GE Healthcare) were immobilized with
approximately 600-800 RU of the respective recombinant antigen
using acetate buffer pH 4.5 according to the manufacturer's manual.
The DLL3.times.CD3 bispecific antibody construct samples were
loaded in a dilution series of the following concentrations: 50 nM,
25 nM, 12.5 nM, 6.25 nM and 3.13 nM diluted in HBS-EP running
buffer (GE Healthcare). Flow rate was 30 .mu.l/min for 3 min, then
HBS-EP running buffer was applied for 8 min to 20 min again at a
flow rate of 30 .mu.l/ml. Regeneration of the chip was performed
using 10 mM glycine 10 mM NaCl pH 1.5 solution. Data sets were
analyzed using BiaEval Software. In general two independent
experiments were performed.
The DLL3.times.CD3 bispecific antibody constructs according to the
invention showed very high affinities to human DLL3 in the
subnanomolar range (with the exception of DLL3-13 having a KD value
in the very low one-digit nanomolar range). Binding to macaque DLL3
was balanced, also showing affinities in similar ranges. The
affinity values as well as the calculated affinity gap are shown in
Table 2.
TABLE-US-00002 TABLE 2 Affinities of DLL3 .times. CD3 bispecific
antibody constructs to human and macaque DLL3 as determined by
Biacore analysis, as well as the calculated interspecies affinity
gaps. DLL3 .times. CD3 bispecific KD hu DLL3 KD cyno DLL3 Affinity
gap antibody construct [nM] [nM] KD mac/KD hu DLL3-4 0.41 0.55 1.34
DLL3-5 0.82 1.03 1.26 DLL3-6 0.55 0.75 1.36 DLL3-7 0.19 0.29 1.52
DLL3-8 0.69 0.96 1.39 DLL3-9 0.35 0.54 1.54 DLL3-10 0.24 0.33 1.38
DLL3-13 1.74 5.55 3.19 DLL3-14 0.47 0.86 1.83 DLL3-15 0.45 0.69
1.53
Furthermore, the binding of the bispecific antibody constructs to
both human CD3 and macaque CD3 was confirmed in a Biacore assay to
be in a low 2-digit nanomolar range (data not shown).
Example 4
Scatchard-Based Analysis of DLL3.times.CD3 Bispecific Antibody
Construct Affinity to Human and Macaque DLL3 on Target Antigen
Positive Cells and Determination of the Interspecies Affinity
Gap
The affinities of DLL3.times.CD3 bispecific antibody constructs to
CHO cells transfected with human or macaque DLL3 were also
determined by Scatchard analysis as the most reliable method for
measuring potential affinity gaps between human and macaque DLL3.
For the Scatchard analysis, saturation binding experiments are
performed using a monovalent detection system to precisely
determine monovalent binding of the DLL3.times.CD3 bispecific
antibody constructs to the respective cell line.
2.times.10.sup.4 cells of the respective cell line (recombinantly
human DLL3-expressing CHO cell line, recombinantly macaque
DLL3-expressing CHO cell line) were incubated each with 50 .mu.l of
a triplet dilution series (twelve dilutions at 1:2) of the
respective DLL3.times.CD3 bispecific antibody construct (until
saturation is reached) starting at 10-20 nM followed by 16 h
incubation at 4.degree. C. under agitation and one residual washing
step. Then, the cells were incubated for another hour with 30 .mu.l
of a CD3.times.ALEXA488 conjugate solution. After one washing step,
the cells were resuspended in 150 .mu.l FACS buffer containing 3.5%
formaldehyde, incubated for further 15 min, centrifuged,
resuspended in FACS buffer and analyzed using a FACS Cantoll
machine and FACS Diva software. Data were generated from two
independent sets of experiments, each using triplicates. Respective
Scatchard analysis was calculated to extrapolate maximal binding
(Bmax). The concentrations of DLL3.times.CD3 bispecific antibody
constructs at half-maximal binding were determined reflecting the
respective KDs. Values of triplicate measurements were plotted as
hyperbolic curves and as S-shaped curves to demonstrate proper
concentration ranges from minimal to optimal binding.
Values depicted in Table 3 were derived from two independent
experiments per DLL3.times.CD3 bispecific antibody construct. Cell
based Scatchard analysis confirmed that the DLL3.times.CD3
bispecific antibody constructs of the invention are subnanomolar in
affinity to human DLL3 and to mac DLL3 and present with a small
cyno/human interspecies affinity gap of around
TABLE-US-00003 TABLE 3 Affinities (KD) of DLL3 .times. CD3
bispecific antibody constructs as determined in cell based
Scatchard analysis with the calculated affinity gap KD macaque
DLL3/KD human DLL3. Antibody constructs were measured in two
independent experiments, each using triplicates. DLL3 .times. CD3
bispecific Cell based Cell based Affinity gap antibody affinity
affinity mac KD construct hu DLL3 [nM] DLL3 [nM] mac/KD hu DLL3-4
0.39 0.24 0.6 DLL3-5 0.33 0.22 0.7 DLL3-6 0.33 0.23 0.7 DLL3-7 0.21
0.33 1.6 DLL3-8 0.18 0.34 1.9 DLL3-9 0.30 0.49 1.6 DLL3-10 0.37
0.32 0.8 DLL3-13 0.24 0.29 1.2 DLL3-14 0.53 0.51 1.0 DLL3-15 0.25
0.50 2.0
Example 5
Bispecific Binding and Interspecies Cross-Reactivity
For confirmation of binding to human DLL3 and CD3 and to cyno DLL3
and CD3, bispecific antibody constructs of the invention were
tested by flow cytometry using CHO cells transfected with human
DLL3, with an artificial human DLL3 isoform (characterized by the
point mutations F172C and L218P), and with macaque DLL3,
respectively, the human DLL3 positive human lung carcinoma cell
line SHP-77, CD3-expressing human T cell leukemia cell line HPB-all
(DSMZ, Braunschweig, ACC483), and the cynomolgus CD3-expressing T
cell line LnPx 4119
For flow cytometry 200,000 cells of the respective cell lines were
incubated for 60 min at 4.degree. C. with 50 .mu.l of purified
bispecific antibody construct at a concentration of 5 .mu.g/ml. The
cells were washed twice in PBS/2% FCS and then incubated with an
in-house mouse antibody (2 .mu.g/ml) specific for the CD3 binding
part of the bispecific antibody constructs for 30 min at 4.degree.
C. After washing, bound mouse antibodies were detected with a goat
anti-mouse Fc.gamma.-PE (1:100) for 30 min at 4.degree. C. Samples
were measured by flow cytometry. Non-transfected CHO cells were
used as negative control.
The results are shown in FIGS. 3A-3C. The DLL3.times.CD3 bispecific
antibody constructs of the invention stained CHO cells transfected
with human DLL3, the artificial DLL3 isoform and with cyno DLL3,
and they also stained the human DLL3 positive human lung carcinoma
cell line SHP-77 (natural expresser). Human and cyno T cell lines
expressing CD3 were also recognized by the bispecific antibody
constructs. Moreover, there was no staining of the negative control
cells (non-transfected CHO, data shown in Example 6).
Example 6
Confirmation of the Absence of Binding to Human Paralogues
Human DLL3 paralogues DLL1 and DLL4 were stably transfected into
CHO cells. The sequences of the paralogues as used in the present
Example are identified in the sequence listing (SEQ ID NOs: 283 and
284). Protein expression was confirmed in FACS analyses with
antibodies specific for the respective paralogues: Antibodies were
anti-human DLL1 MAB1818 (R&D; 5 .mu.g/ml), and anti-human DLL4
MAB1506 (R&D; 5 .mu.g/ml) for DLL4.
The flow cytometry assay was carried out as described in Example 5,
with the exception that bound mouse antibodies were detected with a
goat anti-mouse FITC (1:100). The results are shown in FIG. 4. The
analysis confirmed that none of the DLL3.times.CD3 bispecific
antibody constructs of the invention cross-reacts with the human
DLL3 paralogues DLL1 and DLL4.
Example 7
Identity to Human Germline
In order to analyze the identity/similarity of the sequence of the
antibody constructs to the human antibody germline genes, the DLL3
binding domains of the invention were aligned as follows: Full VL
including all CDRs was aligned; full VH including CDRs 1 and 2 but
except CDR3 was aligned against human antibody germline genes
(Vbase). More details can be found in the specification of this
application. The results are shown in Table 4 below:
TABLE-US-00004 TABLE 4 Identity of VH and VL to human germline DLL3
.times. CD3 bispecific % identity to human germline antibody
construct [VH/VL] DLL3-4 96.9/93.3 DLL3-5 96.9/96.6 DLL3-6
96.9/96.6 DLL3-7 93.9/96.6 DLL3-8 94.8/96.6 DLL3-9 96.9/95.5
DLL3-10 91.9/95.5 DLL3-13 95.9/95.7 DLL3-14 94.9/94.6 DLL3-15
93.9/94.6
Example 8
Cytotoxic Activity
The potency of DLL3.times.CD3 bispecific antibody constructs of the
invention in redirecting effector T cells against DLL3-expressing
target cells was analyzed in five in vitro cytotoxicity assays: The
potency of DLL3.times.CD3 bispecific antibody constructs in
redirecting stimulated human CD8+ effector T cells against human
DLL3-transfected CHO cells was measured in an 18 hour 51-chromium
release assay. The potency of DLL3.times.CD3 bispecific antibody
constructs in redirecting stimulated human CD8+ effector T cells
against the DLL3 positive human lung carcinoma cell line SHP-77 was
measured in an 18 hour 51-chromium release assay. The potency of
DLL3.times.CD3 bispecific antibody constructs in redirecting the T
cells in unstimulated human PBMC against human DLL3-transfected CHO
cells was measured in a 48 hour FACS-based cytotoxicity assay. The
potency of DLL3.times.CD3 bispecific antibody constructs in
redirecting the T cells in unstimulated human PBMC against the
DLL3-positive human cell line SHP-77 was measured in a 48 hour
FACS-based cytotoxicity assay. For confirmation that the
cross-reactive DLL3.times.CD3 bispecific antibody constructs are
capable of redirecting macaque T cells against macaque
DLL3-transfected CHO cells, a 48 hour FACS-based cytotoxicity assay
was performed with a macaque T cell line as effector T cells.
Example 8.1
Chromium Release Assay with Stimulated Human T Cells
Stimulated T cells enriched for CD8.sup.+ T cells were obtained as
described in the following. A petri dish (145 mm diameter, Greiner
bio-one GmbH, Kremsmunster) was coated with a commercially
available anti-CD3 specific antibody (OKT3, Orthoclone) in a final
concentration of 1 .mu.g/ml for 1 hour at 37.degree. C. Unbound
protein was removed by one washing step with PBS.
3-5.times.10.sup.7 human PBMC were added to the precoated petri
dish in 120 ml of RPMI 1640 with stabilized glutamine/10% FCS/IL-2
20 U/ml (Proleukin.RTM., Chiron) and stimulated for 2 days. On the
third day, the cells were collected and washed once with RPMI 1640.
IL-2 was added to a final concentration of 20 U/ml and the cells
were cultured again for one day in the same cell culture medium as
above. CD8.sup.+ cytotoxic T lymphocytes (CTLs) were enriched by
depletion of CD4.sup.+ T cells and CD56.sup.+ NK cells using
Dynal-Beads according to the manufacturer's protocol.
Cyno DLL3- or human DLL3-transfected CHO target cells were washed
twice with PBS and labeled with 11.1 MBq .sup.51Cr in a final
volume of 100 .mu.l RPMI with 50% FCS for 60 minutes at 37.degree.
C. Subsequently, the labeled target cells were washed 3 times with
5 ml RPMI and then used in the cytotoxicity assay. The assay was
performed in a 96-well plate in a total volume of 200 .mu.l
supplemented RPMI with an E:T ratio of 10:1. A starting
concentration of 0.01-1 .mu.g/ml of purified bispecific antibody
construct and threefold dilutions thereof were used. Incubation
time for the assay was 18 hours. Cytotoxicity was determined as
relative values of released chromium in the supernatant relative to
the difference of maximum lysis (addition of Triton-X) and
spontaneous lysis (without effector cells). All measurements were
carried out in quadruplicates. Measurement of chromium activity in
the supernatants was performed in a Wizard 3'' gamma counter
(Perkin Elmer Life Sciences GmbH, Koln, Germany). Analysis of the
results was carried out with Prism 5 for Windows (version 5.0,
GraphPad Software Inc., San Diego, Calif., USA). EC50 values
calculated by the analysis program from the sigmoidal dose response
curves were used for comparison of cytotoxic activity.
Example 8.2
Potency of Redirecting Stimulated Human Effector T Cells Against
Human DLL3-Transfected CHO Cells
The cytotoxic activity of DLL3.times.CD3 bispecific antibody
constructs according to the invention was analyzed in a 51-chromium
(.sup.51Cr) release cytotoxicity assay using CHO cells transfected
with human DLL3 as target cells, and stimulated human CD8.sup.+ T
cells as effector cells. The experiment was carried out as
described in Example 8.1.
The results are shown in Table 5. The DLL3.times.CD3 bispecific
antibody constructs showed very potent cytotoxic activity against
human DLL3 transfected CHO cells in the 1-digit picomolar
range.
TABLE-US-00005 TABLE 5 EC50 values [pM] of DLL3 .times. CD3
bispecific antibody constructs analyzed in a 51-chromium
(.sup.51Cr) release cytotoxicity assay using CHO cells transfected
with human DLL3 as target cells, and stimulated human CD8 T cells
as effector cells. DLL3 .times. CD3 bispecific antibody construct
EC50 [pM] DLL3-4 3.8 DLL3-5 4.2 DLL3-6 2.1 DLL3-7 2.2 DLL3-8 1.2
DLL3-9 1.2 DLL3-10 1.4 DLL3-13 1.8 DLL3-14 5.4 DLL3-15 9.8
Example 8.3
Potency of Redirecting Stimulated Human Effector T Cells Against
the DLL3 Positive Human Lung Carcinoma Cell Line SHP-77
The cytotoxic activity of DLL3.times.CD3 bispecific antibody
constructs was analyzed in a 51-chromium (.sup.51Cr) release
cytotoxicity assay using the DLL3-positive human lung carcinoma
cell line SHP-77 as source of target cells, and stimulated human
CD8.sup.+ T cells as effector cells. The assay was carried out as
described in Example 8.1.
In accordance with the results of the 51-chromium release assays
with stimulated enriched human CD8.sup.+ T lymphocytes as effector
cells and human DLL3-transfected CHO cells as target cells,
DLL3.times.CD3 bispecific antibody constructs of the present
invention are also potent in cytotoxic activity against natural
expresser target cells (see Table 6).
TABLE-US-00006 TABLE 6 EC50 values [pM] of DLL3 .times. CD3
bispecific antibody constructs analyzed in an 18-hour 51-chromium
(.sup.51Cr) release cytotoxicity assay with the DLL3-positive human
lung carcinoma cell line SHP-77 as source of target cells, and
stimulated enriched human CD8 T cells as effector cells. DLL3
.times. CD3 bispecific Row antibody construct EC50 [pM] 1 DLL3-4 27
2 DLL3-5 26 3 DLL3-6 18 4 DLL3-7 23 5 DLL3-8 39 6 DLL3-9 18 7
DLL3-10 31 8 DLL3-13 22 9 DLL3-14 31 10 DLL3-15 36 11 DLL3-18 38 12
DLL3-19 142 13 DLL3-20 171 14 DLL3-21 324 Rows 1-10: Antibody
constructs according to the invention, which bind to a DLL3 epitope
comprised within the region as depicted in SEQ ID NO: 260. (Rows
1-7: Antibody constructs binding to a DLL3 epitope comprised within
the EGF-3 region. Rows 8-10: Antibody constructs binding to a DLL3
epitope comprised within the EGF-4 region.) Rows 11-14: Antibody
constructs binding to a DLL3 epitope which is comprised within the
EGF-5/[EGF-6] region.
Example 8.4
FACS-Based Cytotoxicity Assay with Unstimulated Human PBMC
Isolation of Effector Cells
Human peripheral blood mononuclear cells (PBMC) were prepared by
Ficoll density gradient centrifugation from enriched lymphocyte
preparations (buffy coats), a side product of blood banks
collecting blood for transfusions. Buffy coats were supplied by a
local blood bank and PBMC were prepared on the same day of blood
collection. After Ficoll density centrifugation and extensive
washes with Dulbecco's PBS (Gibco), remaining erythrocytes were
removed from PBMC via incubation with erythrocyte lysis buffer (155
mM NH.sub.4Cl, 10 mM KHCO.sub.3, 100 .mu.M EDTA). Platelets were
removed via the supernatant upon centrifugation of PBMC at
100.times.g. Remaining lymphocytes mainly encompass B and T
lymphocytes, NK cells and monocytes. PBMC were kept in culture at
37.degree. C./5% CO.sub.2 in RPMI medium (Gibco) with 10% FCS
(Gibco).
Depletion of CD14.sup.+ and CD56.sup.+ Cells
For depletion of CD14.sup.+ cells, human CD14 MicroBeads (Milteny
Biotec, MACS, #130-050-201) were used, for depletion of NK cells
human CD56 MicroBeads (MACS, #130-050-401). PBMC were counted and
centrifuged for 10 min at room temperature with 300.times.g. The
supernatant was discarded and the cell pellet resuspended in MACS
isolation buffer [80 .mu.L/10.sup.7 cells; PBS (Invitrogen,
#20012-043), 0.5% (v/v) FBS (Gibco, #10270-106), 2 mM EDTA
(Sigma-Aldrich, # E-6511)]. CD14 MicroBeads and CD56 MicroBeads (20
.mu.L/10.sup.7 cells) were added and incubated for 15 min at
4-8.degree. C. The cells were washed with MACS isolation buffer
(1-2 mL/10.sup.7 cells). After centrifugation (see above),
supernatant was discarded and cells resuspended in MACS isolation
buffer (500 .mu.L/10.sup.8 cells). CD14/CD56 negative cells were
then isolated using LS Columns (Miltenyi Biotec, #130-042-401).
PBMC w/o CD14.sup.+/CD56.sup.+ cells were cultured in RPMI complete
medium i.e. RPMI1640 (Biochrom AG, # FG1215) supplemented with 10%
FBS (Biochrom AG, #+S0115), 1.times. non-essential amino acids
(Biochrom AG, # K0293), 10 mM Hepes buffer (Biochrom AG, # L1613),
1 mM sodium pyruvate (Biochrom AG, # L0473) and 100 U/mL
penicillin/streptomycin (Biochrom AG, # A2213) at 37.degree. C. in
an incubator until needed.
Target Cell Labeling
For the analysis of cell lysis in flow cytometry assays, the
fluorescent membrane dye DiOC.sub.18 (DiO) (Molecular Probes, #
V22886) was used to label human DLL3- or macaque DLL3-transfected
CHO cells as target cells and distinguish them from effector cells.
Briefly, cells were harvested, washed once with PBS and adjusted to
10.sup.6 cell/mL in PBS containing 2% (v/v) FBS and the membrane
dye DiO (5 .mu.L/10.sup.6 cells). After incubation for 3 min at
37.degree. C., cells were washed twice in complete RPMI medium and
the cell number adjusted to 1.25.times.10.sup.5 cells/mL. The
vitality of cells was determined using 0.5% (v/v) isotonic EosinG
solution (Roth, #45380).
Flow Cytometry Based Analysis
This assay was designed to quantify the lysis of cyno or human
DLL3-transfected CHO cells in the presence of serial dilutions of
DLL3 bispecific antibody constructs. Equal volumes of DiO-labeled
target cells and effector cells (i.e., PBMC w/o CD14.sup.+ cells)
were mixed, resulting in an E:T cell ratio of 10:1. 160 .mu.l of
this suspension were transferred to each well of a 96-well plate.
40 .mu.L of serial dilutions of the DLL3.times.CD3 bispecific
antibody constructs and a negative control bispecific (a CD3-based
bispecific antibody construct recognizing an irrelevant target
antigen) or RPMI complete medium as an additional negative control
were added. The bispecific antibody-mediated cytotoxic reaction
proceeded for 48 hours in a 7% CO.sub.2 humidified incubator. Then
cells were transferred to a new 96-well plate and loss of target
cell membrane integrity was monitored by adding propidium iodide
(PI) at a final concentration of 1 .mu.g/mL. PI is a membrane
impermeable dye that normally is excluded from viable cells,
whereas dead cells take it up and become identifiable by
fluorescent emission.
Samples were measured by flow cytometry on a FACSCanto II
instrument and analyzed by FACSDiva software (both from Becton
Dickinson). Target cells were identified as DiO-positive cells.
PI-negative target cells were classified as living target cells.
Percentage of cytotoxicity was calculated according to the
following formula:
.times..times..times..times..times..times..times..times.
##EQU00001## .times..times..times..times. ##EQU00001.2##
Using GraphPad Prism 5 software (Graph Pad Software, San Diego),
the percentage of cytotoxicity was plotted against the
corresponding bispecific antibody construct concentrations. Dose
response curves were analyzed with the four parametric logistic
regression models for evaluation of sigmoid dose response curves
with fixed hill slope and EC50 values were calculated.
Example 8.5
Potency of Redirecting Unstimulated Human PBMC Against Human
DLL3-Transfected CHO Cells
The cytotoxic activity of DLL3.times.CD3 bispecific antibody
constructs was analyzed in a FACS-based cytotoxicity assay using
CHO cells transfected with human DLL3 as target cells, and
unstimulated human PBMC as effector cells. The assay was carried
out as described in Example 8.4 above.
The results of the FACS-based cytotoxicity assays with unstimulated
human PBMC as effector cells and human DLL3-transfected CHO cells
as targets are shown in Table 7.
TABLE-US-00007 TABLE 7 EC50 values [pM] of DLL3 .times. CD3
bispecific antibody constructs as measured in a 48-hour FACS-based
cytotoxicity assay with unstimulated human PBMC as effector cells
and CHO cells transfected with human DLL3 as target cells. DLL3
.times. CD3 bispecific Row antibody construct EC50 [pM] 1 DLL3-4 53
2 DLL3-5 36 3 DLL3-6 44 4 DLL3-7 40 5 DLL3-8 43 6 DLL3-9 43 7
DLL3-10 40 8 DLL3-13 116 9 DLL3-14 66 10 DLL3-15 57 11 DLL3-18 169
12 DLL3-19 107 13 DLL3-20 171 14 DLL3-21 85 Rows 1-10: Antibody
constructs according to the invention, which bind to a DLL3 epitope
comprised within the region as depicted in SEQ ID NO: 260. (Rows
1-7: Antibody constructs binding to a DLL3 epitope comprised within
the EGF-3 region. Rows 8-10: Antibody constructs binding to a DLL3
epitope comprised within the EGF-4 region.) Rows 11-14: Antibody
constructs binding to a DLL3 epitope which is comprised within the
EGF-5/[EGF-6] region.
Expectedly, EC50 values were generally higher in cytotoxicity
assays with unstimulated PBMC as effector cells compared with
cytotoxicity assays using stimulated human CD8.sup.+ T cells (see
Example 8.2).
Example 8.6
Potency of Redirecting Unstimulated Human PBMC Against the
DLL3-Positive Lung Carcinoma Cell Line SHP-77
The cytotoxic activity of DLL3.times.CD3 bispecific antibody
constructs was furthermore analyzed in a FACS-based cytotoxicity
assay using the DLL3-positive human lung carcinoma cell line SHP-77
as a source of target cells and unstimulated human PBMC as effector
cells. The assay was carried out as described in Example 8.4 above.
The results are shown in Table 8.
TABLE-US-00008 TABLE 8 EC50 values [pM] of DLL3 .times. CD3
bispecific antibody constructs as measured in a 48-hour FACS-based
cytotoxicity assay with unstimulated human PBMC as effector cells
and the human cell line SHP-77 as source of target cells. DLL3
.times. CD3 bispecific Row antibody construct EC50 [pM] 1 DLL3-4 44
2 DLL3-5 65 3 DLL3-6 31 4 DLL3-7 30 5 DLL3-8 24 6 DLL3-9 33 7
DLL3-10 32 8 DLL3-13 49 9 DLL3-14 65 10 DLL3-15 66 11 DLL3-18 76 12
DLL3-19 180 13 DLL3-20 1540 14 DLL3-21 770 Rows 1-10: Antibody
constructs according to the invention, which bind to a DLL3 epitope
comprised within the region as depicted in SEQ ID NO: 260. (Rows
1-7: Antibody constructs binding to a DLL3 epitope comprised within
the EGF-3 region. Rows 8-10: Antibody constructs binding to a DLL3
epitope comprised within the EGF-4 region.) Rows 11-14: Antibody
constructs binding to a DLL3 epitope which is comprised within the
EGF-5/[EGF-6] region.
Example 8.7
Potency of Redirecting Macaque T Cells Against Macaque
DLL3-Expressing CHO Cells
Finally, the cytotoxic activity of DLL3.times.CD3 bispecific
antibody constructs was analyzed in a FACS-based cytotoxicity assay
using CHO cells transfected with macaque (cyno) DLL3 as target
cells, and the macaque T cell line 4119LnPx (Knappe et al. Blood
95:3256-61 (2000)) as source of effector cells. Target cell
labeling of macaque DLL3-transfected CHO cells and flow cytometry
based analysis of cytotoxic activity was performed as described
above.
Results are shown in Table 9. Macaque T cells from cell line
4119LnPx were induced to efficiently kill macaque DLL3-transfected
CHO cells by DLL3.times.CD3 bispecific antibody constructs
according to the invention. The antibody constructs presented
potently with 2-digit picomolar EC50-values in this assay,
confirming that they are very active in the macaque system.
TABLE-US-00009 TABLE 9 EC50 values [pM] of DLL3 .times. CD3
bispecific antibody constructs as measured in a 48-hour FACS-based
cytotoxicity assay with macaque T cell line 4119LnPx as effector
cells and CHO cells transfected with macaque DLL3 as target cells.
DLL3 .times. CD3 bispecific Row antibody construct EC50 [pM] 1
DLL3-4 36 2 DLL3-5 42 3 DLL3-6 40 4 DLL3-7 101 5 DLL3-8 44 6 DLL3-9
58 7 DLL3-10 42 8 DLL3-13 65 9 DLL3-14 28 10 DLL3-15 32 11 DLL3-18
134 12 DLL3-19 66 13 DLL3-20 231 14 DLL3-21 86 Rows 1-10: Antibody
constructs according to the invention, which bind to a DLL3 epitope
comprised within the region as depicted in SEQ ID NO: 260. (Rows
1-7: Antibody constructs binding to a DLL3 epitope comprised within
the EGF-3 region. Rows 8-10: Antibody constructs binding to a DLL3
epitope comprised within the EGF-4 region.) Rows 11-14: Antibody
constructs binding to a DLL3 epitope which is comprised within the
EGF-5/[EGF-6] region.
Example 9
Monomer to Dimer Conversion after (i) Three Freeze/Thaw Cycles and
(ii) 7 Days of Incubation at 250 .mu.g/Ml
Bispecific DLL3.times.CD3 antibody monomeric construct were
subjected to different stress conditions followed by high
performance SEC to determine the percentage of initially monomeric
antibody construct, which had been converted into dimeric antibody
construct.
(i) 25 .mu.g of monomeric antibody construct were adjusted to a
concentration of 250 .mu.g/ml with generic formulation buffer and
then frozen at -80.degree. C. for 30 min followed by thawing for 30
min at room temperature. After three freeze/thaw cycles the dimer
content was determined by HP-SEC.
(ii) 25 .mu.g of monomeric antibody construct were adjusted to a
concentration of 250 .mu.g/ml with generic formulation buffer
followed by incubation at 37.degree. C. for 7 days. The dimer
content was determined by HP-SEC.
A high resolution SEC Column TSK Gel G3000 SWXL (Tosoh,
Tokyo-Japan) was connected to an Akta Purifier 10 FPLC (GE
Lifesciences) equipped with an A905 Autosampler. Column
equilibration and running buffer consisted of 100 mM KH2PO4-200 mM
Na2SO4 adjusted to pH 6.6. The antibody solution (25 .mu.g protein)
was applied to the equilibrated column and elution was carried out
at a flow rate of 0.75 ml/min at a maximum pressure of 7 MPa. The
whole run was monitored at 280, 254 and 210 nm optical absorbance.
Analysis was done by peak integration of the 210 nm signal recorded
in the Akta Unicorn software run evaluation sheet. Dimer content
was calculated by dividing the area of the dimer peak by the total
area of monomer plus dimer peak.
The results are shown in Table 10 below. The DLL3.times.CD3
bispecific antibody constructs of the invention presented with
dimer percentages of 0.0% after three freeze/thaw cycles, and with
dimer percentages of .ltoreq.2% after 7 days of incubation at
37.degree. C.
TABLE-US-00010 TABLE 10 Percentage of monomeric versus dimeric DLL3
.times. CD3 bispecific antibody constructs as determined by High
Performance Size Exclusion Chromatography (HP-SEC). DLL3 .times.
CD3 Percentage of dimer Percentage of dimer bispecific antibody
after three after 7 days of construct freeze/thaw cycles incubation
at 37.degree. C. DLL3-4 0.7 0.0 DLL3-5 1.5 0.0 DLL3-6 1.3 0.0
DLL3-7 1.2 0.0 DLL3-8 1.5 0.0 DLL3-9 1.8 0.0 DLL3-10 0.6 0.0
DLL3-13 1.6 0.0 DLL3-14 0.4 0.0 DLL3-15 1.2 0.0
Example 10
Thermostability
Antibody aggregation temperature was determined as follows: 40
.mu.l of antibody construct solution at 250 .mu.g/ml were
transferred into a single use cuvette and placed in a Wyatt Dynamic
Light Scattering device DynaPro Nanostar (Wyatt). The sample was
heated from 40.degree. C. to 70.degree. C. at a heating rate of
0.5.degree. C./min with constant acquisition of the measured
radius. Increase of radius indicating melting of the protein and
aggregation was used by the software package delivered with the DLS
device to calculate the aggregation temperature of the antibody
construct.
All tested DLL3.times.CD3 bispecific antibody constructs of the
invention showed thermal stability with aggregation temperatures
.gtoreq.45.degree. C., as shown in Table 11 below. The group of
antibody constructs binding to an epitope of DLL3 which is
comprised within the EGF-4 region (as depicted in SEQ ID NO: 259)
even had a thermal stability of .gtoreq.50.degree. C., more
precisely, of .gtoreq.54.degree. C.
TABLE-US-00011 TABLE 11 Thermostability of the bispecific antibody
constructs as determined by DLS (dynamic light scattering) DLL3
.times. CD3 bispecific Thermostability antibody construct (DLS
.degree. C. aggregation) DLL3-4 59.3 DLL3-5 45.4 DLL3-6 58.8 DLL3-7
58.2 DLL3-8 49.8 DLL3-9 49.6 DLL3-10 52.9 DLL3-13 54.0 DLL3-14 57.0
DLL3-15 56.3
Example 11
Stability after Incubation for 24 Hours in Human Plasma
Purified bispecific antibody constructs were incubated at a ratio
of 1:5 in a human plasma pool at 37.degree. C. for 96 hours at a
final concentration of 2-20 .mu.g/ml. After plasma incubation the
antibody constructs were compared in a 51-chromium release assay
with stimulated enriched human CD8+ T cells and human
DLL3-transfected CHO cells at a starting concentration of 0.01-0.1
.mu.g/ml and with an effector to target cell (E:T) ratio of 10:1
(assay as described in Example 8.1). Non-incubated, freshly thawed
bispecific antibody constructs were included as controls.
The results are shown in Table 12 below; exemplary results for the
two antibody constructs DLL-4 and DLL-14 are also shown in FIG. 5.
All tested antibody constructs had a very favourable plasma
stability (EC50 plasma/EC50 control) of .ltoreq.2.5. The group of
antibody constructs binding to an epitope of DLL3 which is
comprised within the EGF-4 region (as depicted in SEQ ID NO: 259)
even had a plasma stability of .ltoreq.1.5, more precisely, of
.ltoreq.1.1.
TABLE-US-00012 TABLE 12 EC50 values of the antibody constructs with
and without plasma incubation and calculated plasma/control value
DLL3 .times. CD3 Plasma to bispecific Control ratio antibody
EC.sub.50 [pM] (EC.sub.50 plasma/EC.sub.50 construct w/plasma w/o
plasma control) DLL3-4 3.6 3.8 0.9 DLL3-5 5.4 4.2 1.3 DLL3-6 4.8
2.1 2.3 DLL3-7 2.7 2.2 1.2 DLL3-8 1.0 1.2 0.8 DLL3-9 1.2 1.2 1.0
DLL3-10 2.5 1.4 1.8 DLL3-13 2.0 1.8 1.1 DLL3-14 4.8 5.4 0.9 DLL3-15
7.8 9.8 0.8
Example 12
Turbidity at 2500 .mu.g/Ml Antibody Concentration
1 ml of purified antibody construct solution of a concentration of
250 .mu.g/ml was concentrated by spin concentration units to 2500
.mu.g/ml. After 16 h storage at 5.degree. C. the turbidity of the
antibody solution was determined by OD340 nm optical absorption
measurement against the generic formulation buffer.
The results are shown in Table 13 below. All tested antibody
constructs have a very favourable turbidity of .ltoreq.0.1, with
the exception of one construct with a turbidity slightly above 0.1.
The group of antibody constructs binding to an epitope of DLL3
which is comprised within the EGF-4 region (as depicted in SEQ ID
NO: 259) even have a turbidity of .ltoreq.0.08.
TABLE-US-00013 TABLE 13 Turbidity of the antibody constructs after
concentration to 2.5 mg/ml over night DLL3 .times. CD3 bispecific
Turbidity at 2500 .mu.g/ml antibody construct [OD340] DLL3-4 0.073
DLL3-5 0.106 DLL3-6 0.080 DLL3-7 0.089 DLL3-8 0.069 DLL3-9 0.085
DLL3-10 0.091 DLL3-13 0.075 DLL3-14 0.073 DLL3-15 0.078
Example 13
Protein Homogeneity by High Resolution Cation Exchange
Chromatography
The protein homogeneity the antibody constructs of the invention
was analyzed by high resolution cation exchange chromatography
CIEX.
50 .mu.g of antibody construct monomer were diluted with 50 ml
binding buffer A (20 mM sodium dihydrogen phosphate, 30 mM NaCl,
0.01% sodium octanate, pH 5.5), and 40 ml of this solution were
applied to a 1 ml BioPro SP-F column (YMC, Germany) connected to an
Akta Micro FPLC device (GE Healthcare, Germany). After sample
binding, a wash step with further binding buffer was carried out.
For protein elution, a linear increasing salt gradient using buffer
B (20 mM sodium dihydrogen phosphate, 1000 mM NaCl, 0.01% sodium
octanate, pH 5.5) up to 50% percent buffer B was applied over 10
column volumes. The whole run was monitored at 280, 254 and 210 nm
optical absorbance. Analysis was done by peak integration of the
280 nm signal recorded in the Akta Unicorn software run evaluation
sheet.
The results are shown in Table 14 below. All tested antibody
constructs have a very favourable homogeneity of .gtoreq.80% (area
under the curve (=AUC) of the main peak), The group of antibody
constructs binding to an epitope of DLL3 which is comprised within
the EGF-3 region (as depicted in SEQ ID NO: 258) even have a
homogeneity of .gtoreq.90%.
TABLE-US-00014 TABLE 14 Protein homogeneity of the antibody
constructs (% AUC of main peak) DLL3 .times. CD3 bispecific Protein
homogeneity antibody construct % AUC of main peak DLL3-4 96 DLL3-5
100 DLL3-6 95 DLL3-7 93 DLL3-8 100 DLL3-9 93 DLL3-10 90 DLL3-13 100
DLL3-14 100 DLL3-15 83
Example 14
Surface hydrophobicity as measured by HIC Butyl
The surface hydrophobicity of bispecific antibody constructs of the
invention was tested in Hydrophobic Interaction Chromatography HIC
in flow-through mode.
50 .mu.g of antibody construct monomer were diluted with generic
formulation buffer to a final volume of 500 .mu.l (10 mM citric
acid, 75 mM lysine HCl, 4% trehalose, pH 7.0) and applied to a 1 ml
Butyl Sepharose FF column (GE Healthcare, Germany) connected to a
Akta Purifier FPLC system (GE Healthcare, Germany). The whole run
was monitored at 280, 254 and 210 nm optical absorbance. Analysis
was done by peak integration of the 280 nm signal recorded in the
Akta Unicorn software run evaluation sheet. Elution behavior was
evaluated by comparing area and velocity of rise and decline of
protein signal thereby indicating the strength of interaction of
the BiTE albumin fusion with the matrix.
The antibody constructs had a good elution behaviour, which was
mostly rapid and complete.
Example 15
Potency Gap Between the Monomeric and the Dimeric Isoform of
Bispecific Antibody Constructs
In order to determine the difference in cytotoxic activity between
the monomeric and the dimeric isoform of individual DLL3.times.CD3
bispecific antibody constructs (referred to as potency gap), an 18
hour 51-chromium release cytotoxicity assay was carried out as
described hereinabove (Example 8.1) with purified bispecific
antibody construct monomer and dimer. Effector cells were
stimulated enriched human CD8+ T cells. Target cells were hu DLL3
transfected CHO cells. Effector to target cell (E:T) ratio was
10:1. The potency gap was calculated as ratio between EC50
values.
The results are shown in Table 15 below; exemplary results for the
two antibody constructs DLL-4 and DLL-14 are also shown in FIG. 6.
Potency gaps of the tested DLL3.times.CD3 bispecific antibody
constructs were between 0.2 and 1.0. There is hence no
substantially more active dimer compared to its respective
monomer.
TABLE-US-00015 TABLE 15 Potency gap between the monomeric and the
dimeric isoform DLL3 .times. CD3 bispecific antibody EC.sub.50 [pM]
EC.sub.50 [pM] Ratio construct monomer dimer EC.sub.50
monomer/EC.sub.50 dimer DLL3-4 3.8 5.7 0.7 DLL3-5 4.2 11 0.4 DLL3-6
2.1 13 0.2 DLL3-7 2.2 4.2 0.5 DLL3-8 1.2 3.4 0.4 DLL3-9 1.2 3.8 0.3
DLL3-10 1.4 1.4 1.0 DLL3-13 1.8 3.0 0.6 DLL3-14 5.4 8.7 0.6 DLL3-15
9.8 25 0.4
Example 16
In Vitro Internalization Assay
Changes in the potency of the DLL3.times.CD3 bispecific antibody
construct as a function of preincubation of the construct on the
target cells in the absence of T cells were measured. If the
antibody construct is internalized, it should undergo lysosomal
degradation. The effective concentration should decrease with time,
and thus the apparent potency should decrease as well. The effect
is observed with other targets, for which this is a known
phenomenon, but no impact was observed with the DLL3.times.CD3
bispecific antibody construct. The assay was carried out as
follows:
T cells were counted and diluted to a concentration of
1.times.10.sup.5/ml in assay media. SHP-77 cells were counted and
plated at 2500 cells per well (cpw). The antibody construct was
diluted serially 1:2 (with Bravo), at a starting concentration of
100 nM. The antibody construct was added to the culture assay
plates to allow for 0 hours, 1 hour or 2 hours of incubation prior
to addition of the T cells. Then the T cells were plated at 25000
cpw, and the assay was incubated for 48 hours at 37.degree. C.
SHP-77 cell survival was analyzed with the Steady-Glo.RTM. system
(25 .mu.l/well). The results are shown in FIG. 7 and suggest no
significant internalization of the antibody construct
DLL3-4.times.CD3 (I2C).
Example 17
Shedding Study
In order to analyze whether the cytotoxic activity of the
DLL3.times.CD3 bispecific antibody constructs of the invention is
significantly impaired by the presence of shed DLL, the following
assay was carried out: T cells were counted and diluted to a
concentration of 1.times.10.sup.5/ml in assay media. SHP-77 cells
were counted and diluted to a concentration of
1.25.times.10.sup.5/ml in assay media with increasing
concentrations of soluble DLL3 between 0.3 nM and 12 nM. SHP-77
cells were plated at 2500 cells per well (cpw), and T cells were
added at 25000 cpw. The antibody construct was diluted serially 1:2
(with Bravo), and added to the culture assay (with Bravo).
Incubation occurred for 48 hours at 37.degree. C. SHP-77 cell
survival was analyzed with the Steady-Glo.RTM. system (25
.mu.l/well).
Example 18
Mouse Xenograft Efficacy Study
The anti-tumor activity of an HLE DLL3.times.CD3 bispecific
antibody construct (SEQ ID NO: 517) was tested in a model of female
NOD/SCID mice which were subcutaneously injected on day 1 of the
study with 5.times.10.sup.6 human DLL3 positive SCLC (SHP-77 luc)
or 5.times.10.sup.6 human DLL3 positive melanoma (WM 266-4) cells.
Effector cells (2.times.10.sup.7 in vitro expanded and activated
living human CD3+ T cells) were injected intraperitoneally on day
12. Treatment start was on day 16 (WM 266-4) or on day 18 (SHP-77
luc). The antibody construct was administered four times every five
days (q5d.times.4) by i.v. bolus injections. The treatment groups
were as follows: SCLC model (SHP-77 luc)/7 mice per group
Vehicle-treated group with T cells DLL3.times.CD3 bispecific
antibody construct: 10 mg/kg per administration Melanoma model
(WM266-4)/9 mice per group Vehicle-treated group with T cells
DLL3.times.CD3 bispecific antibody construct: 10 mg/kg per
administration DLL3.times.CD3 bispecific antibody construct: 2
mg/kg per administration
Tumors were measured by caliper during the study and progress
evaluated by intergroup comparison of tumor volumes (TV). The tumor
growth inhibition T/C [%] on day x is determined by calculating the
tumor volume as T/C (%)=100.times. (median TV of analyzed
group)/(median TV of control group), and the calculated values are
depicted in the following table 16:
TABLE-US-00016 TABLE 16 T/C values of the mouse xenograft studies
with SHP-77 luc cells and WM266-4 cells T/C (%) T/C (%) T/C (%) Day
of SHP-77 model WM266-4 model WM266-4 model Study 10 mg/kg 10 mg/kg
2 mg/kg 15 103 101 101 17 92 76 77 20 64 42 47 23 50 36 38 26 52 31
35 29 34 46 40 31 33 56 55 33 30 64 75 36 24 n.a n.a 38 19 n.a
n.a
The results are furthermore shown in FIGS. 8A and 8B. Significant
tumor growth inhibition was shown in both tumor models and at both
tested dose levels of 2 and 10 mg/kg.
Example 19
Cyno Exploratory Toxicology Study
An exploratory toxicology study was carried out with a non
half-life extended DLL3.times.CD3 bispecific antibody construct
(SEQ ID NO: 554). Three female cynomolgus monkeys were dosed via
continuous i.v. infusion for 16 days (5, 15, and 45 .mu.g/kg/day
for 3 days each, followed by 100 .mu.g/kg/day for 7 days). No test
article-related clinical observations, changes in body temperature,
food consumption or body weight were observed.
Consistent with expectations for the pharmacology of a
DLL3.times.CD3 bispecific antibody construct, circulating T
lymphocyte populations (total T-lymphocytes, T-helper and
T-cytotoxic lymphocytes, NK cells, B-lymphocytes, and CD25+
activated T-lymphocytes) were decreased on the first day of dosing
and remained lower throughout the duration of the study in all
animals. Activation markers (CD69 and CD25 on activated T cells)
were increased on day 1, but not at later time points in the
study.
To summarize, the DLL3.times.CD3 bispecific antibody construct was
very well tolerated even at the highest dose tested (100
.mu.g/kg/d, .gtoreq.300.times.EC.sub.50).
Example 20
Cyno PK Studys
A cyno PK study was performed with naive male cynomolgus monkeys.
Three different albumin-fused DLL3.times.CD3 (I2C) bispecific
antibody constructs (DLL3-4, DLL3-6 and DLL3-14) were administered
as i.v. single bolus at a concentration of 12 .mu.g/kg. For each of
the molecules a group of two animals was used.
A separate cyno PK study was performed under the same conditions,
but with DLL3-4 in different Fc fusion versions.
Blood samples were collected pre-dose and at 0.05, 0.5, 1, 4, 8,
24, 48, 72, 120, 168, 240, and 336 hours post-dose. Serum was
prepared for determination of serum concentrations of the molecules
in an immunoassay. The assay was performed by capturing the
antibody constructs via their target moiety, while an antibody
directed against the CD3-binding part of the construct was used for
detection. The serum concentration-time profiles were used to
determine PK parameters. The pharmacokinetic parameters were
determined using standard non-compartmental analysis (NCA) methods.
The following PK parameters were assessed: AUC.sub.inf (Area under
the serum concentration-time curve), V.sub.ss (volume of
distribution at steady state), CL (systemic clearance) and terminal
t.sub.1/2 (terminal half-life). For all antibody constructs, serum
levels were quantifiable for all time points in all animals after
their administration. No clinical observations were made in any of
the treated animals.
The pharmacokinetics of the tested antibody constructs are shown in
FIGS. 9A and 9B, and the PK parameters are summarized as mean of
n=2 in table 17 below.
The albumin-fused constructs showed a favorable PK profile
consistent with a once or twice weekly dosing schedule in a human
patient. An even more favorable PK profile supporting once weekly
dosing or even every other week dosing was observed with an Fc
fusion construct (scFc).
TABLE-US-00017 TABLE 17 Pharmacokinetic parameters of HLE variants
of DLL3 .times. CD3 bispecific antibody constructs in cynomolgus
monkeys AUC.sub.inf V.sub.ss CL t.sub.1/2 Antibody construct [ng *
h/mL] [mL/kg] [mL/h/kg] [h] DLL3-4 .times. I2C HALB 20,669 70 0.58
98 DLL3-6 .times. I2C HALB 20,228 67 0.59 103 DLL3-14 .times. I2C
HALB 21,597 107 0.55 154 DLL3-4 .times. I2C scFc 29,746 118 0.40
213 DLL3-4 (cc) .times. I2C scFc 24,769 144 0.48 234 DLL3-4 .times.
I2C hetero Fc 14,639 166 0.82 173
TABLE-US-00018 TABLE 18 SEQ ID DLL3 Desig- Format/ NO epitope
nation Source Amino acid sequence 1 DLL3-1 VH CDR1 DYGIH 2 DLL3-1
VH CDR2 VISYHGSNKYYARSVKG 3 DLL3-1 VH CDR3 EIPFGMDV 4 DLL3-1 VL
CDR1 RSSQSLLHSDGYNYLD 5 DLL3-1 VL CDR2 LGSNRAS 6 DLL3-1 VL CDR3
MQALQTPLT 7 DLL3-1 VH
QVQLVESGGGVVQSGRSLRLSCAASGFTFSDYGIHWVRQAPGKGLEWVAVISYHGSNKYYA- R
SVKGRFTVSRDNSKNTLYLQMNSLRAEDTAVYYCAREIPFGMDVWGQGTTVTVSS 8 DLL3-1 VL
DIVMTQTPLSLPVTPGEPASISCRSSQSLLHSDGYNYLDWYLQKPGQSPQLLIYLGSNRAS- G
VPDRFSGSGSGTDFTLTISRVEAEDVGVYYCMQALQTPLTFGGGTKVDIK 9 N-term DLL3-1
scFv QVQLVESGGGVVQSGRSLRLSCAASGFTFSDYGIHWVRQAPGKGLEWVAVISY-
HGSNKYYAR
SVKGRFTVSRDNSKNTLYLQMNSLRAEDTAVYYCAREIPFGMDVWGQGTTVTVSSGGGGSGG
GGSGGGGSDIVMTQTPLSLPVTPGEPASISCRSSQSLLHSDGYNYLDWYLQKPGQSPQLLIY
LGSNRASGVPDRFSGSGSGTDFTLTISRVEAEDVGVYYCMQALQTPLTFGGGTKVDIK 10
DLL3-1 bispecific
QVQLVESGGGVVQSGRSLRLSCAASGFTFSDYGIHWVRQAPGKGLEWVAVIS- YHGSNKYYAR
xI2C molecule
SVKGRFTVSRDNSKNTLYLQMNSLRAEDTAVYYCAREIPFGMDVWGQGTTVTVSSGGG- GSGG
GGSGGGGSDIVMTQTPLSLPVTPGEPASISCRSSQSLLHSDGYNYLDWYLQKPGQSPQLLIY
LGSNRASGVPDRFSGSGSGTDFTLTISRVEAEDVGVYYCMQALQTPLTFGGGTKVDIKSGGG
GSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYAT
YYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVT
VSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQ
APRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKL TVL
11 DLL3-2 VH CDR1 GYYMH 12 DLL3-2 VH CDR2 WINPNSGDTNYAQKFQG 13
DLL3-2 VH CDR3 DANIAALDAFEI 14 DLL3-2 VL CDR1 RASQSISSYLN 15 DLL3-2
VL CDR2 AASSLQS 16 DLL3-2 VL CDR3 QQSYSTPLT 17 DLL3-2 VH
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGDTNY- AQ
KFQGRVTMTRDTSISTAYMELSRLTSDDTAVYYCARDANIAALDAFEIWGQGTMVTVSS 18
DLL3-2 VL
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPS- RF
SGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIK 19 N-term. DLL3-2
scFv QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWI-
NPNSGDTNYAQ
KFQGRVTMTRDTSISTAYMELSRLTSDDTAVYYCARDANIAALDAFEIWGQGTMVTVSSGGG
GSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIK 20 DLL3-2
bispecific QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWIN-
PNSGDTNYAQ xI2C molecule
KFQGRVTMTRDTSISTAYMELSRLTSDDTAVYYCARDANIAALDAFEIWGQGTMVTVS- SGGG
GSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIKSGGGG
SEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATY
YADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTV
SSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQA
PRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLT VL
21 DLL3-3 VH CDR1 SYGMH 22 DLL3-3 VH CDR2 VISYHGRDTYYARSVKG 23
DLL3-3 VH CDR3 DGATVTSYYYSGMDV 24 DLL3-3 VL CDR1 RASQGISNYLA 25
DLL3-3 VL CDR2 LASSLQS 26 DLL3-3 VL CDR3 QQYNFYPFT 27 DLL3-3 VH
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYHGRDTYY- AR
SVKGRFTISRDNSKNTLYLHMNSLRAEDTAVYYCARDGATVTSYYYSGMDVWGQGTTVTVSS K 28
DLL3-3 VL
DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWFQQKPGKAPKSLIYLASSLQSGVPS- KF
SGSGSGTDFTLTISSLQPEDFATYYCQQYNFYPFTFGPGTKVDIK 29 EGF-1 DLL3-3 scFv
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISY- HGRDTYYAR
SVKGRFTISRDNSKNTLYLHMNSLRAEDTAVYYCARDGATVTSYYYSGMDVWGQGTTVTVSS
GGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWFQQKPGKAPKSL
IYLASSLQSGVPSKFSGSGSGTDFTLTISSLQPEDFATYYCQQYNFYPFTFGPGTKVDIK 30
DLL3-3 bispecific
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIS- YHGRDTYYAR
xI2C molecule
SVKGRFTISRDNSKNTLYLHMNSLRAEDTAVYYCARDGATVTSYYYSGMDVWGQGTTV- TVSS
GGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWFQQKPGKAPKSL
IYLASSLQSGVPSKFSGSGSGTDFTLTISSLQPEDFATYYCQQYNFYPFTFGPGTKVDIKSG
GGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNY
ATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTL
VTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKP
GQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGT
KLTVL 31 DLL3-4 VH CDR1 SYYWS 32 DLL3-4 VH CDR2 YVYYSGTTNYNPSLKS 33
DLL3-4 VH CDR3 IAVTGFYFDY 34 DLL3-4 VL CDR1 RASQRVNNNYLA 35 DLL3-4
VL CDR2 GASSRAT 36 DLL3-4 VL CDR3 QQYDRSPLT 37 DLL3-4 VH
QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGYVYYSGTTNYN- PS
LKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASIAVTGFYFDYWGQGTLVTVSS 38 DLL3-4
VL EIVLTQSPGTLSLSPGERVTLSCRASQRVNNNYLAWYQQRPGQAPRLLIYGASSRATGIP- DR
FSGSGSGTDFTLTISRLEPEDFAVYYCQQYDRSPLTFGGGTKLEIK 39 EGF-3 DLL3-4 scFv
QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGYVYY- SGTTNYNPS
LKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASIAVTGFYFDYWGQGTLVTVSSGGGGSG
GGGSGGGGSEIVLTQSPGTLSLSPGERVTLSCRASQRVNNNYLAWYQQRPGQAPRLLIYGAS
SRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDRSPLTFGGGTKLEIK 40 DLL3-4
bispecific QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGYVY-
YSGTTNYNPS xI2C molecule
LKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASIAVTGFYFDYWGQGTLVTVSSGG- GGSG
GGGSGGGGSEIVLTQSPGTLSLSPGERVTLSCRASQRVNNNYLAWYQQRPGQAPRLLIYGAS
SRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDRSPLTFGGGTKLEIKSGGGGSE
VQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYA
DSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSS
GGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPR
GLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 41
DLL3-5 VH CDR1 SYYWS 42 DLL3-5 VH CDR2 YIYYSGRTNYYPSLKS 43 DLL3-5
VH CDR3 IAVAGFFFDY 44 DLL3-5 VL CDR1 RASQSVNKNYLA 45 DLL3-5 VL CDR2
GASSRAT 46 DLL3-5 VL CDR3 QQYDRSPLT 47 DLL3-5 VH
QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGYIYYSGRTNYY- PS
LKSRVTISIDTSKNQFSLKLSSVTAADTAVYYCARIAVAGFFFDYWGQGTLVTVSS 48 DLL3-5
VL EIVLTQSPGTLSLSPGERATLSCRASQSVNKNYLAWYQQKPGQAPRLLIYGASSRATGIP- DR
FSGSGSGTDFTLTISRLEPEDFAVYYCQQYDRSPLTFGGGTKLEIK 49 EGF-3 DLL3-5 scFv
QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGYIYY- SGRTNYYPS
LKSRVTISIDTSKNQFSLKLSSVTAADTAVYYCARIAVAGFFFDYWGQGTLVTVSSGGGGSG
GGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVNKNYLAWYQQKPGQAPRLLIYGAS
SRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDRSPLTFGGGTKLEIK 50 DLL3-5
bispecific QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGYIY-
YSGRTNYYPS xI2C molecule
LKSRVTISIDTSKNQFSLKLSSVTAADTAVYYCARIAVAGFFFDYWGQGTLVTVSSGG- GGSG
GGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVNKNYLAWYQQKPGQAPRLLIYGAS
SRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDRSPLTFGGGTKLEIKSGGGGSE
VQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYA
DSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSS
GGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPR
GLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 51
DLL3-6 VH CDR1 SFYWS 52 DLL3-6 VH CDR2 YIYYSGTTNYNPSLKS 53 DLL3-6
VH CDR3 IAVAGFFFDY 54 DLL3-6 VL CDR1 RASQSVNKNYLA 55 DLL3-6 VL CDR2
GASSRAT 56 DLL3-6 VL CDR3 QQYDRSPLT 57 DLL3-6 VH
QVQLQESGPGLVKPSETLSLTCTVSGASISSFYWSWIRQPPGKGLEWIGYIYYSGTTNYN- PS
LKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARIAVAGFFFDYWGQGTLVTVSS 58 DLL3-6
VL EIVLTQSPGTLSLSPGERATLSCRASQSVNKNYLAWYQQKPGQAPRLLIYGASSRATGIP- DR
FSGSGSGTDFTLTISRLEPEDFAVYYCQQYDRSPLTFGGGTKVEIK 59 EGF-3 DLL3-6 scFv
QVQLQESGPGLVKPSETLSLTCTVSGASISSFYWSWIRQPPGKGLEWIGYIYY- SGTTNYNPS
LKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARIAVAGFFFDYWGQGTLVTVSSGGGGSG
GGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVNKNYLAWYQQKPGQAPRLLIYGAS
SRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDRSPLTFGGGTKVEIK 60 DLL3-6
bispecific QVQLQESGPGLVKPSETLSLTCTVSGASISSFYWSWIRQPPGKGLEWIGYIY-
YSGTTNYNPS xI2C molecule
LKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARIAVAGFFFDYWGQGTLVTVSSGG- GGSG
GGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVNKNYLAWYQQKPGQAPRLLIYGAS
SRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDRSPLTFGGGTKVEIKSGGGGSE
VQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYA
DSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSS
GGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPR
GLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 61
DLL3-7 VH CDR1 SFYWS 62 DLL3-7 VH CDR2 YIYYSGTTNYNPSLKS 63 DLL3-7
VH CDR3 IAVAGFFFDY 64 DLL3-7 VL CDR1 RASQSVNKNYLA 65 DLL3-7 VL CDR2
GASSRAT 66 DLL3-7 VL CDR3 QQYDRSPLT 67 DLL3-7 VH
QVQLQESGPGLVKPSQTLSLTCTVSGASISSFYWSWIRQPPGKGLEWIGYIYYSGTTNYN- PS
LKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARIAVAGFFFDYWGQGTLVTVSS 68 DLL3-7
VL
EIVLTQSPGTLSLSPGERATLSCRASQSVNKNYLAWYQQKPGQAPRLLIYGASSRATGIP-
DR FSGSGSGTDFTLTISRLEPEDFAVYYCQQYDRSPLTFGGGTKVEIK 69 EGF-3 DLL3-7
scFv QVQLQESGPGLVKPSQTLSLTCTVSGASISSFYWSWIRQPPGKGLEWIGYIYY-
SGTTNYNPS
LKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARIAVAGFFFDYWGQGTLVTVSSGGGGSG
GGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVNKNYLAWYQQKPGQAPRLLIYGAS
SRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDRSPLTFGGGTKVEIK 70 DLL3-7
bispecific QVQLQESGPGLVKPSQTLSLTCTVSGASISSFYWSWIRQPPGKGLEWIGYIY-
YSGTTNYNPS xI2C molecule
LKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARIAVAGFFFDYWGQGTLVTVSSGG- GGSG
GGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVNKNYLAWYQQKPGQAPRLLIYGAS
SRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDRSPLTFGGGTKVEIKSGGGGSE
VQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYA
DSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSS
GGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPR
GLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 71
DLL3-8 VH CDR1 SFYWS 72 DLL3-8 VH CDR2 YIYYSGTTNYNPSLKS 73 DLL3-8
VH CDR3 IAVAGFFFDY 74 DLL3-8 VL CDR1 RASQSVNKNYLA 75 DLL3-8 VL CDR2
GASSRAT 76 DLL3-8 VL CDR3 QQYDRSPLT 77 DLL3-8 VH
QVQLQEWGPGLVKPSETLSLTCTVSGASISSFYWSWIRQPPGKGLEWIGYIYYSGTTNYN- PS
LKSRVTISVDTSKNQLSLKLSSVTAADTAVYYCARIAVAGFFFDYWGQGTLVTVSSK 78 DLL3-8
VL EIVLTQSPGTLSLSPGERATLSCRASQSVNKNYLAWYQQKPGQAPRLLIYGASSRATGIP- DR
FSGSGSGTDFTLTISRLEPEDFAVYYCQQYDRSPLTFGGGTKVDIK 79 EGF-3 DLL3-8 scFv
QVQLQEWGPGLVKPSETLSLTCTVSGASISSFYWSWIRQPPGKGLEWIGYIYY- SGTTNYNPS
LKSRVTISVDTSKNQLSLKLSSVTAADTAVYYCARIAVAGFFFDYWGQGTLVTVSSGGGGSG
GGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVNKNYLAWYQQKPGQAPRLLIYGAS
SRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDRSPLTFGGGTKVDIK 80 DLL3-8
bispecific QVQLQEWGPGLVKPSETLSLTCTVSGASISSFYWSWIRQPPGKGLEWIGYIY-
YSGTTNYNPS xI2C molecule
LKSRVTISVDTSKNQLSLKLSSVTAADTAVYYCARIAVAGFFFDYWGQGTLVTVSSGG- GGSG
GGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVNKNYLAWYQQKPGQAPRLLIYGAS
SRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDRSPLTFGGGTKVDIKSGGGGSE
VQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYA
DSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSS
GGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPR
GLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 81
DLL3-9 VH CDR1 SFYWS 82 DLL3-9 VH CDR2 YIYYSGTTNYNPSLKS 83 DLL3-9
VH CDR3 IAVAGFFFDY 84 DLL3-9 VL CDR1 RASQSVNKNYLA 85 DLL3-9 VL CDR2
GASSRAT 86 DLL3-9 VL CDR3 QQYDRSPLT 87 DLL3-9 VH
QVQLQESGPGLVKPSETLSLTCTVSGASISSFYWSWIRQPPGKGLEWIGYIYYSGTTNYN- PS
LKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARIAVAGFFFDYWGQGTLVTVSS 88 DLL3-9
VL EIVLTQSPGTLSLSPGESATLSCRASQSVNKNYLAWYQQKPGQAPRLLIYGASSRATGIP- DR
FSGSGSGTDFTLTISRLEPEDFAVYYCQQYDRSPLTFGGGTRLEIK 89 EGF-3 DLL3-9 scFv
QVQLQESGPGLVKPSETLSLTCTVSGASISSFYWSWIRQPPGKGLEWIGYIYY- SGTTNYNPS
LKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARIAVAGFFFDYWGQGTLVTVSSGGGGSG
GGGSGGGGSEIVLTQSPGTLSLSPGESATLSCRASQSVNKNYLAWYQQKPGQAPRLLIYGAS
SRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDRSPLTFGGGTRLEIK 90 DLL3-9
bispecific QVQLQESGPGLVKPSETLSLTCTVSGASISSFYWSWIRQPPGKGLEWIGYIY-
YSGTTNYNPS xI2C molecule
LKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARIAVAGFFFDYWGQGTLVTVSSGG- GGSG
GGGSGGGGSEIVLTQSPGTLSLSPGESATLSCRASQSVNKNYLAWYQQKPGQAPRLLIYGAS
SRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDRSPLTFGGGTRLEIKSGGGGSE
VQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYA
DSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSS
GGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPR
GLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 91
DLL3-10 VH CDR1 SYYWS 92 DLL3-10 VH CDR2 YIFYNGITNYNPSLKS 93
DLL3-10 VH CDR3 IHSGSFSFDY 94 DLL3-10 VL CDR1 RASQSVSRGYLA 95
DLL3-10 VL CDR2 GASSRAT 96 DLL3-10 VL CDR3 QQYDTSPIT 97 DLL3-10 VH
QVQLQESGPGLVKPSQTLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGYIFYNGITNY- NPS
LKSRVTISLDTSKNQFSLKLSSVTAADTAKYYCARIHSGSFSFDYWDQGTLVTVSS 98 DLL3-10
VL EIVMTQSPGTLSLSPGERATLSCRASQSVSRGYLAWYQQKPGQAPRLLIYGASSRATDI- PDR
FSGSGSGTDFTLTISRLEPEDFAVYYCQQYDTSPITFGQGTKVEIK 99 EGF-3 DLL3-10
scFv QVQLQESGPGLVKPSQTLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGYIF-
YNGITNYNPS
LKSRVTISLDTSKNQFSLKLSSVTAADTAKYYCARIHSGSFSFDYWDQGTLVTVSSGGGGSG
GGGSGGGGSEIVMTQSPGTLSLSPGERATLSCRASQSVSRGYLAWYQQKPGQAPRLLIYGAS
SRATDIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDTSPITFGQGTKVEIK 100 DLL3-10
bispecific QVQLQESGPGLVKPSQTLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGY-
IFYNGITNYNPS xI2C molecule
LKSRVTISLDTSKNQFSLKLSSVTAADTAKYYCARIHSGSFSFDYWDQGTLVTVSSGG- GGSG
GGGSGGGGSEIVMTQSPGTLSLSPGERATLSCRASQSVSRGYLAWYQQKPGQAPRLLIYGAS
SRATDIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDTSPITFGQGTKVEIKSGGGGSE
VQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYA
DSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSS
GGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPR
GLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 101
DLL3-11 VH CDR1 NAGMS 102 DLL3-11 VH CDR2 RIKNKIDGGTTDFAAPVKG 103
DLL3-11 VH CDR3 RGWYGDYFDY 104 DLL3-11 VL CDR1 RSSQSLLHSNGYNYLD 105
DLL3-11 VL CDR2 LGSNRAS 106 DLL3-11 VL CDR3 MQALQTPFT 107 DLL3-11
VH EVQLVESGGGLVKPGGSLRLSCAASGFIFNNAGMSWVRQAPGKGLEWVGRIKNKIDGG- TTDF
AAPVKGRFTISRDDSKNTLYLQMNSLKAEDTAVYYCTARGWYGDYFDYWGQGTLVTVSS 108
DLL3-11 VL
DIVMTQTPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSN- RASG
VPDRFSGSGSGTDFTLKISRVEAEDVGIYYCMQALQTPFTFGPGTKVEIK 109 EGF-3
DLL3-11 scFv EVQLVESGGGLVKPGGSLRLSCAASGFIFNNAGMSWVRQAPGKGLEWVGRI-
KNKIDGGTTDF
AAPVKGRFTISRDDSKNTLYLQMNSLKAEDTAVYYCTARGWYGDYFDYWGQGTLVTVSSGGG
GSGGGGSGGGGSDIVMTQTPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQ
LLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCMQALQTPFTFGPGTKVEIK 110
DLL3-11 bispecific
EVQLVESGGGLVKPGGSLRLSCAASGFIFNNAGMSWVRQAPGKGLEWVGR- IKNKIDGGTTDF
xI2C molecule
AAPVKGRFTISRDDSKNTLYLQMNSLKAEDTAVYYCTARGWYGDYFDYWGQGTLVTVS- SGGG
GSGGGGSGGGGSDIVMTQTPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQ
LLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCMQALQTPFTFGPGTKVEIK
SGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYN
NYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQG
TLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ
KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGG
GTKLTVL 111 DLL3-12 VH CDR1 SYDIH 112 DLL3-12 VH CDR2
VISSHGSNKNYARSVKG 113 DLL3-12 VH CDR3 DGYSGNDPFYYYYHGMDV 114
DLL3-12 VL CDR1 RASQSISSYLN 115 DLL3-12 VL CDR2 AASSLQS 116 DLL3-12
VL CDR3 QQSFTTPLT 117 DLL3-12 VH
QVQLVESGGGVVQPGRSLRLSCAASGFSFSSYDIHWVRQAPGKGLEWVAVISSHGSNK- NYAR
SVKGRFTISRDNSKNTLYLQMNSLKAEDTAVYYCARDGYSGNDPFYYYYHGMDVWGQGTTVT VSS
118 DLL3-12 VL
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGV- PSRF
SGSGSGTDFSLTISSLQPEDFATYYCQQSFTTPLTFGGGTKVEIK 119 EGF-3/[4] DLL3-12
scFv QVQLVESGGGVVQPGRSLRLSCAASGFSFSSYDIHWVRQAPGKGLEW-
VAVISSHGSNKNYAR
SVKGRFTISRDNSKNTLYLQMNSLKAEDTAVYYCARDGYSGNDPFYYYYHGMDVWGQGTTVT
VSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAP
KLLIYAASSLQSGVPSRFSGSGSGTDFSLTISSLQPEDFATYYCQQSFTTPLTFGGGTKVEI K
120 DLL3-12 bispecific
QVQLVESGGGVVQPGRSLRLSCAASGFSFSSYDIHWVRQAPGKGLEWVAV- ISSHGSNKNYAR
xI2C molecule
SVKGRFTISRDNSKNTLYLQMNSLKAEDTAVYYCARDGYSGNDPFYYYYHGMDVWGQG- TTVT
VSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAP
KLLIYAASSLQSGVPSRFSGSGSGTDFSLTISSLQPEDFATYYCQQSFTTPLTFGGGTKVEI
KSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKY
NNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQ
GTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQ
QKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFG
GGTKLTVL 121 DLL3-13 VH CDR1 SYYMH 122 DLL3-13 VH CDR2
IINPSDGSTNYAQNFQG 123 DLL3-13 VH CDR3 GGNSAFYSYYDMDV 124 DLL3-13 VL
CDR1 RSSQSLVYRDGNTYLS 125 DLL3-13 VL CDR2 KVSNWQS 126 DLL3-13 VL
CDR3 MQGTHWPPT 127 DLL3-13 VH
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSDGST- NYAQ
NFQGRVTMTRDTSTNTVYMELSSLRSEDTAVYYCARGGNSAFYSYYDMDVWGQGTTVTVSS 128
DLL3-13 VL
DVVMTQSPLSLPVTLGQPASISCRSSQSLVYRDGNTYLSWFQQRPGQSPRRLIYKVSN- WQSG
VPDRFSGSGSGTDFTLKISRVEAEDVGVYFCMQGTHWPPTFGQGTKVEIK 129 EGF-4
DLL3-13 scFv QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGII-
NPSDGSTNYAQ
NFQGRVTMTRDTSTNTVYMELSSLRSEDTAVYYCARGGNSAFYSYYDMDVWGQGTTVTVSSG
GGGSGGGGSGGGGSDVVMTQSPLSLPVTLGQPASISCRSSQSLVYRDGNTYLSWFQQRPGQS
PRRLIYKVSNWQSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCMQGTHWPPTFGQGTKVE IK
130 DLL3-13 bispecific
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGI- INPSDGSTNYAQ
xI2C molecule
NFQGRVTMTRDTSTNTVYMELSSLRSEDTAVYYCARGGNSAFYSYYDMDVWGQGTTVT- VSSG
GGGSGGGGSGGGGSDVVMTQSPLSLPVTLGQPASISCRSSQSLVYRDGNTYLSWFQQRPGQS
PRRLIYKVSNWQSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCMQGTHWPPTFGQGTKVE
IKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSK
YNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWG
QGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWV
QQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVF
GGGTKLTVL 131 DLL3-14 VH CDR1 NYYMH 132 DLL3-14 VH CDR2
IINPSDGSTSYAQKFQG
133 DLL3-14 VH CDR3 GGNSAFYSYYDMDV 134 DLL3-14 VL CDR1
RSSQSLVYRDGNTYLS 135 DLL3-14 VL CDR2 KVSNWQS 136 DLL3-14 VL CDR3
MQGTHWPPT 137 DLL3-14 VH
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGLGLEWMGIINPSDGST- SYAQ
KFQGRVTMTRDTSTNTVYMDLSSLRSEDTAVYYCARGGNSAFYSYYDMDVWGQGTTVTVSS 138
DLL3-14 VL
DVVMTQTPLSLPVTLGQPASISCRSSQSLVYRDGNTYLSWFQQRPGQSPRRLIYKVSN- WQSG
VPDRFSGGGSGTDFTLKISRVEAEDVGVYYCMQGTHWPPTFGQGTKVEIK 139 EGF-4
DLL3-14 scFv QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGLGLEWMGII-
NPSDGSTSYAQ
KFQGRVTMTRDTSTNTVYMDLSSLRSEDTAVYYCARGGNSAFYSYYDMDVWGQGTTVTVSSG
GGGSGGGGSGGGGSDVVMTQTPLSLPVTLGQPASISCRSSQSLVYRDGNTYLSWFQQRPGQS
PRRLIYKVSNWQSGVPDRFSGGGSGTDFTLKISRVEAEDVGVYYCMQGTHWPPTFGQGTKVE IK
140 DLL3-14 bispecific
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGLGLEWMGI- INPSDGSTSYAQ
xI2C molecule
KFQGRVTMTRDTSTNTVYMDLSSLRSEDTAVYYCARGGNSAFYSYYDMDVWGQGTTVT- VSSG
GGGSGGGGSGGGGSDVVMTQTPLSLPVTLGQPASISCRSSQSLVYRDGNTYLSWFQQRPGQS
PRRLIYKVSNWQSGVPDRFSGGGSGTDFTLKISRVEAEDVGVYYCMQGTHWPPTFGQGTKVE
IKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSK
YNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWG
QGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWV
QQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVF
GGGTKLTVL 141 DLL3-15 VH CDR1 GYYIH 142 DLL3-15 VH CDR2
IINPSDGSTSYGQNFQG 143 DLL3-15 VH CDR3 GGNSAFYSYYDMDV 144 DLL3-15 VL
CDR1 RSSQSLAYRDGNTYLS 145 DLL3-15 VL CDR2 KVSNWQS 146 DLL3-15 VL
CDR3 MQGTHWPPT 147 DLL3-15 VH
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYIHWVRQAPGQGLEWMGIINPSDGST- SYGQ
NFQGRVTMTRDTSTNTVYMELSSLRSEDTAVYYCARGGNSAFYSYYDMDVWGQGTTVTVSS 148
DLL3-15 VL
DVVMTQSPLSLPVTLGQPASISCRSSQSLAYRDGNTYLSWFQQRPGQSPRRLIYKVSN- WQSG
VPDRFSGSGSGTDFTLKISRVEAEDVGVYFCMQGTHWPPTFGQGTKVEIK 149 EGF-4
DLL3-15 scFv QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYIHWVRQAPGQGLEWMGII-
NPSDGSTSYGQ
NFQGRVTMTRDTSTNTVYMELSSLRSEDTAVYYCARGGNSAFYSYYDMDVWGQGTTVTVSSG
GGGSGGGGSGGGGSDVVMTQSPLSLPVTLGQPASISCRSSQSLAYRDGNTYLSWFQQRPGQS
PRRLIYKVSNWQSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCMQGTHWPPTFGQGTKVE IK
150 DLL3-15 bispecific
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYIHWVRQAPGQGLEWMGI- INPSDGSTSYGQ
xI2C molecule
NFQGRVTMTRDTSTNTVYMELSSLRSEDTAVYYCARGGNSAFYSYYDMDVWGQGTTVT- VSSG
GGGSGGGGSGGGGSDVVMTQSPLSLPVTLGQPASISCRSSQSLAYRDGNTYLSWFQQRPGQS
PRRLIYKVSNWQSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCMQGTHWPPTFGQGTKVE
IKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSK
YNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWG
QGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWV
QQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVF
GGGTKLTVL 151 DLL3-16 VH CDR1 GHYMH 152 DLL3-16 VH CDR2
IINPSDGSTNYAQKFQG 153 DLL3-16 VH CDR3 GTTVVHYSYYDMDV 154 DLL3-16 VL
CDR1 RSSQSLVYRDGNTYLT 155 DLL3-16 VL CDR2 KVSNWQS 156 DLL3-16 VL
CDR3 MQGTHWPPT 157 DLL3-16 VH
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGHYMHWVRQAPGQGLEWMGIINPSDGST- NYAQ
KFQGRVTMTRDTSTSTVYMELRSLRSEDTAVYYCTRGTTVVHYSYYDMDVWGQGTTVTVSS 158
DLL3-16 VL
DVVMTQTPLSLPVTLGQPASISCRSSQSLVYRDGNTYLTWFQQRPGQSPRRLIYKVSN- WQSG
VPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPPTFGGGTKVEIK 159 EGF-4
DLL3-16 scFv QVQLVQSGAEVKKPGASVKVSCKASGYTFTGHYMHWVRQAPGQGLEWMGII-
NPSDGSTNYAQ
KFQGRVTMTRDTSTSTVYMELRSLRSEDTAVYYCTRGTTVVHYSYYDMDVWGQGTTVTVSSG
GGGSGGGGSGGGGSDVVMTQTPLSLPVTLGQPASISCRSSQSLVYRDGNTYLTWFQQRPGQS
PRRLIYKVSNWQSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPPTFGGGTKVE IK
160 DLL3-16 bispecific
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGHYMHWVRQAPGQGLEWMGI- INPSDGSTNYAQ
xI2C molecule
KFQGRVTMTRDTSTSTVYMELRSLRSEDTAVYYCTRGTTVVHYSYYDMDVWGQGTTVT- VSSG
GGGSGGGGSGGGGSDVVMTQTPLSLPVTLGQPASISCRSSQSLVYRDGNTYLTWFQQRPGQS
PRRLIYKVSNWQSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPPTFGGGTKVE
IKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSK
YNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWG
QGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWV
QQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVF
GGGTKLTVL 161 DLL3-17 VH CDR1 NYFMH 162 DLL3-17 VH CDR2
IINPSDGSTSYAQNFQG 163 DLL3-17 VH CDR3 GGNSAFYSYYDMDV 164 DLL3-17 VL
CDR1 RSSQSLVYRDGNTYLS 165 DLL3-17 VL CDR2 RVSNWQS 166 DLL3-17 VL
CDR3 MQGTYWPPT 167 DLL3-17 VH
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYFMHWVRQAPGLGLEWMGIINPSDGST- SYAQ
NFQGRVTMTRDTSTNTVYMELSSLRSEDTAVYYCARGGNSAFYSYYDMDVWGQGTTVTVSS 168
DLL3-17 VL
DVVMTQSPLSLPVTLGQPASISCRSSQSLVYRDGNTYLSWFQQRPGQSPRRLIYRVSN- WQSG
VPDRFSGSGSGTDFTLKISRVEAEDVGVYFCMQGTYWPPTFGQGTKVDIK 169 EGF-4
DLL3-17 scFv QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYFMHWVRQAPGLGLEWMGII-
NPSDGSTSYAQ
NFQGRVTMTRDTSTNTVYMELSSLRSEDTAVYYCARGGNSAFYSYYDMDVWGQGTTVTVSSG
GGGSGGGGSGGGGSDVVMTQSPLSLPVTLGQPASISCRSSQSLVYRDGNTYLSWFQQRPGQS
PRRLIYRVSNWQSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCMQGTYWPPTFGQGTKVD IK
170 DLL3-17 bispecific
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYFMHWVRQAPGLGLEWMGI- INPSDGSTSYAQ
xI2C molecule
NFQGRVTMTRDTSTNTVYMELSSLRSEDTAVYYCARGGNSAFYSYYDMDVWGQGTTVT- VSSG
GGGSGGGGSGGGGSDVVMTQSPLSLPVTLGQPASISCRSSQSLVYRDGNTYLSWFQQRPGQS
PRRLIYRVSNWQSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCMQGTYWPPTFGQGTKVD
IKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSK
YNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWG
QGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWV
QQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVF
GGGTKLTVL 171 DLL3-18 VH CDR1 NYGMH 172 DLL3-18 VH CDR2
VISHHGSSKYYARSVKG 173 DLL3-18 VH CDR3 DWWELVFDY 174 DLL3-18 VL CDR1
KSSQSLLHSDGKTFLY 175 DLL3-18 VL CDR2 EVSNRFS 176 DLL3-18 VL CDR3
LQGIHLPFT 177 DLL3-18 VH
QVQLVESGGGAVQPGRSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVAVISHHGSSK- YYAR
SVKGRFTISRDNSKNTLYLEMNSLRAEDTAVYYCARDWWELVFDYWGQGTLVTVSS 178
DLL3-18 VL
DIVMTQTPLSLSVTPGQPASISCKSSQSLLHSDGKTFLYWYLQKPGQPPQLLIYEVSN- RFSG
VPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQGIHLPFTFGPGTKVEIK 179 EGF-5/[6]
DLL3-18 scFv QVQLVESGGGAVQPGRSLRLSCAASGFTFSNYGMHWVRQAPGKGLEW-
VAVISHHGSSKYYAR
SVKGRFTISRDNSKNTLYLEMNSLRAEDTAVYYCARDWWELVFDYWGQGTLVTVSSGGGGSG
GGGSGGGGSDIVMTQTPLSLSVTPGQPASISCKSSQSLLHSDGKTFLYWYLQKPGQPPQLLI
YEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQGIHLPFTFGPGTKVEIK 180
DLL3-18 bispecific
QVQLVESGGGAVQPGRSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVAV- ISHHGSSKYYAR
xI2C molecule
SVKGRFTISRDNSKNTLYLEMNSLRAEDTAVYYCARDWWELVFDYWGQGTLVTVSSGG- GGSG
GGGSGGGGSDIVMTQTPLSLSVTPGQPASISCKSSQSLLHSDGKTFLYWYLQKPGQPPQLLI
YEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQGIHLPFTFGPGTKVEIKSGG
GGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYA
TYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLV
TVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPG
QAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTK LTVL
181 DLL3-19 VH CDR1 NSRMGVS 182 DLL3-19 VH CDR2 HIFSNDGKSYSTSLKS
183 DLL3-19 VH CDR3 YNYDSSGYYYSFFDY 184 DLL3-19 VL CDR1 RASQSISSYLN
185 DLL3-19 VL CDR2 AASSLQS 186 DLL3-19 VL CDR3 QQGYSSPFT 187
DLL3-19 VH
QVTLKESGPMLVKPTETLTLTCTVSGFSLSNSRMGVSWIRQPPGRALEWLAHIFSNDG- KSYS
TSLKSRLTISKDTSKSQVVLTMTNMDPVDTATYYCARYNYDSSGYYYSFFDYWGQGTLVTVS S
188 DLL3-19 VL
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGV- PSRF
SGSGSGTDFTLTISSLQPEDFATYYCQQGYSSPFTFGGGTKVEIK 189 EGF-5/[6] DLL3-19
scFv QVTLKESGPMLVKPTETLTLTCTVSGFSLSNSRMGVSWIRQPPGRAL-
EWLAHIFSNDGKSYS
TSLKSRLTISKDTSKSQVVLTMTNMDPVDTATYYCARYNYDSSGYYYSFFDYWGQGTLVTVS
SGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKL
LIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYSSPFTFGGGTKVEIK 190
DLL3-19 bispecific
QVTLKESGPMLVKPTETLTLTCTVSGFSLSNSRMGVSWIRQPPGRALEWL- AHIFSNDGKSYS
xI2C molecule
TSLKSRLTISKDTSKSQVVLTMTNMDPVDTATYYCARYNYDSSGYYYSFFDYWGQGTL- VTVS
SGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKL
LIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYSSPFTFGGGTKVEIKS
GGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNN
YATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGT
LVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQK
PGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGG
TKLTVL 191 DLL3-20 VH CDR1 NARMGVS 192 DLL3-20 VH CDR2
HIFSTDEKSYSTSLKS 193 DLL3-20 VH CDR3 YYYDSSGYYYSFFDY 194 DLL3-20 VL
CDR1 RASQSIRSYLN 195 DLL3-20 VL CDR2 GASNLQS 196 DLL3-20 VL CDR3
QQSYSSPFT 197 DLL3-20 VH
QVTLKESGPVLVKPTETLTLTCTVSGFSLSNARMGVSWLRQPPGKALEWLAHIFSTDE- KSYS
TSLKSRLTISKDTSKSQVVLTMTNMDPVDTATYYCARYYYDSSGYYYSFFDYWGQGTLVTVS S
198 DLL3-20 VL
DIQMTQSPSSLSASVGDRVTITCRASQSIRSYLNWYQQKPGKAPKLLIYGASNLQSGV- PSRF
SGSGSGTDFTLTISSLQPEDFATYYCQQSYSSPFTFGGGTKVEIK
199 EGF-5/[6] DLL3-20 scFv
QVTLKESGPVLVKPTETLTLTCTVSGFSLSNARMGVSWLRQPPGKAL- EWLAHIFSTDEKSYS
TSLKSRLTISKDTSKSQVVLTMTNMDPVDTATYYCARYYYDSSGYYYSFFDYWGQGTLVTVS
SGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSIRSYLNWYQQKPGKAPKL
LIYGASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSSPFTFGGGTKVEIK 200
DLL3-20 bispecific
QVTLKESGPVLVKPTETLTLTCTVSGFSLSNARMGVSWLRQPPGKALEWL- AHIFSTDEKSYS
xI2C molecule
TSLKSRLTISKDTSKSQVVLTMTNMDPVDTATYYCARYYYDSSGYYYSFFDYWGQGTL- VTVS
SGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSIRSYLNWYQQKPGKAPKL
LIYGASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSSPFTFGGGTKVEIKS
GGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNN
YATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGT
LVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQK
PGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGG
TKLTVL 201 DLL3-21 VH CDR1 SYYIH 202 DLL3-21 VH CDR2
IINPSGGSKSYAQKFRG 203 DLL3-21 VH CDR3 SMSTVTSDAFDI 204 DLL3-21 VL
CDR1 RASQSISNYLN 205 DLL3-21 VL CDR2 AASSLQS 206 DLL3-21 VL CDR3
QQSYSAPLT 207 DLL3-21 VH
QVQLVQSGAEVKKPGASVKVSCKASGYAFTSYYIHWVRQAPGQGLEWMGIINPSGGSK- SYAQ
KFRGRVTMTRDTSTSTVYMELSSLTSEDTAVYYCARSMSTVTSDAFDIWGQGTMVTVSS 208
DLL3-21 VL
DIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQKPGKAPKLLIYAASSLQSGV- PSRF
SGSGSGTEFTLTISSLQPEDFATYYCQQSYSAPLTFGGGTKVDIK 209 EGF-5/[6] DLL3-21
scFv QVQLVQSGAEVKKPGASVKVSCKASGYAFTSYYIHWVRQAPGQGLEW-
MGIINPSGGSKSYAQ
KFRGRVTMTRDTSTSTVYMELSSLTSEDTAVYYCARSMSTVTSDAFDIWGQGTMVTVSSGGG
GSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQSYSAPLTFGGGTKVDIK 210
DLL3-21 bispecific
QVQLVQSGAEVKKPGASVKVSCKASGYAFTSYYIHWVRQAPGQGLEWMGI- INPSGGSKSYAQ
xI2C molecule
KFRGRVTMTRDTSTSTVYMELSSLTSEDTAVYYCARSMSTVTSDAFDIWGQGTMVTVS- SGGG
GSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQSYSAPLTFGGGTKVDIKSGGGG
SEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATY
YADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTV
SSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQA
PRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLT VL
211 EGF-3 DLL3-4 bispecific
QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLE- WIGYVYYSGTTNYNPS
xF12Q molecule
LKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASIAVTGFYFDYWGQGTLVTVSSG- GGGSG
GGGSGGGGSEIVLTQSPGTLSLSPGERVTLSCRASQRVNNNYLAWYQQRPGQAPRLLIYGAS
SRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDRSPLTFGGGTKLEIKSGGGGSE
VQLVESGGGLVQPGGSLRLSCAASGFTFNSYAMNWVRQAPGKGLEWVARIRSKYNNYATYYA
DSVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWWAYWGQGTLVTVSS
GGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPR
GLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 212
EGF-3 DLL3-5 bispecific
QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLE- WIGYIYYSGRTNYYPS
xF12Q molecule
LKSRVTISIDTSKNQFSLKLSSVTAADTAVYYCARIAVAGFFFDYWGQGTLVTVSSG- GGGSG
GGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVNKNYLAWYQQKPGQAPRLLIYGAS
SRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDRSPLTFGGGTKLEIKSGGGGSE
VQLVESGGGLVQPGGSLRLSCAASGFTFNSYAMNWVRQAPGKGLEWVARIRSKYNNYATYYA
DSVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWWAYWGQGTLVTVSS
GGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPR
GLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 213
EGF-3 DLL3-6 bispecific
QVQLQESGPGLVKPSETLSLTCTVSGASISSFYWSWIRQPPGKGLE- WIGYIYYSGTTNYNPS
xF12Q molecule
LKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARIAVAGFFFDYWGQGTLVTVSSG- GGGSG
GGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVNKNYLAWYQQKPGQAPRLLIYGAS
SRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDRSPLTFGGGTKVEIKSGGGGSE
VQLVESGGGLVQPGGSLRLSCAASGFTFNSYAMNWVRQAPGKGLEWVARIRSKYNNYATYYA
DSVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWWAYWGQGTLVTVSS
GGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPR
GLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 214
EGF-3 DLL3-7 bispecific
QVQLQESGPGLVKPSQTLSLTCTVSGASISSFYWSWIRQPPGKGLE- WIGYIYYSGTTNYNPS
xF12Q molecule
LKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARIAVAGFFFDYWGQGTLVTVSSG- GGGSG
GGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVNKNYLAWYQQKPGQAPRLLIYGAS
SRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDRSPLTFGGGTKVEIKSGGGGSE
VQLVESGGGLVQPGGSLRLSCAASGFTFNSYAMNWVRQAPGKGLEWVARIRSKYNNYATYYA
DSVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWWAYWGQGTLVTVSS
GGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPR
GLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 215
EGF-3 DLL3-8 bispecific
QVQLQEWGPGLVKPSETLSLTCTVSGASISSFYWSWIRQPPGKGLE- WIGYIYYSGTTNYNPS
xF12Q molecule
LKSRVTISVDTSKNQLSLKLSSVTAADTAVYYCARIAVAGFFFDYWGQGTLVTVSSG- GGGSG
GGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVNKNYLAWYQQKPGQAPRLLIYGAS
SRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDRSPLTFGGGTKVDIKSGGGGSE
VQLVESGGGLVQPGGSLRLSCAASGFTFNSYAMNWVRQAPGKGLEWVARIRSKYNNYATYYA
DSVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWWAYWGQGTLVTVSS
GGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPR
GLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 216
EGF-3 DLL3-9 bispecific
QVQLQESGPGLVKPSETLSLTCTVSGASISSFYWSWIRQPPGKGLE- WIGYIYYSGTTNYNPS
xF12Q molecule
LKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARIAVAGFFFDYWGQGTLVTVSSG- GGGSG
GGGSGGGGSEIVLTQSPGTLSLSPGESATLSCRASQSVNKNYLAWYQQKPGQAPRLLIYGAS
SRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDRSPLTFGGGTRLEIKSGGGGSE
VQLVESGGGLVQPGGSLRLSCAASGFTFNSYAMNWVRQAPGKGLEWVARIRSKYNNYATYYA
DSVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWWAYWGQGTLVTVSS
GGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPR
GLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 217
EGF-3 DLL3-10 bispecific
QVQLQESGPGLVKPSQTLSLTCTVSGGSISSYYWSWIRQPPGKGL- EWIGYIFYNGITNYNPS
xF12Q molecule
LKSRVTISLDTSKNQFSLKLSSVTAADTAKYYCARIHSGSFSFDYWDQGTLVTVSSG- GGGSG
GGGSGGGGSEIVMTQSPGTLSLSPGERATLSCRASQSVSRGYLAWYQQKPGQAPRLLIYGAS
SRATDIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDTSPITFGQGTKVEIKSGGGGSE
VQLVESGGGLVQPGGSLRLSCAASGFTFNSYAMNWVRQAPGKGLEWVARIRSKYNNYATYYA
DSVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWWAYWGQGTLVTVSS
GGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPR
GLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 218
EGF-4 DLL3-13 bispecific
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGL- EWMGIINPSDGSTNYAQ
xF12Q molecule
NFQGRVTMTRDTSTNTVYMELSSLRSEDTAVYYCARGGNSAFYSYYDMDVWGQGTTV- TVSSG
GGGSGGGGSGGGGSDVVMTQSPLSLPVTLGQPASISCRSSQSLVYRDGNTYLSWFQQRPGQS
PRRLIYKVSNWQSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCMQGTHWPPTFGQGTKVE
IKSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFNSYAMNWVRQAPGKGLEWVARIRSK
YNNYATYYADSVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWWAYWG
QGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWV
QQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVF
GGGTKLTVL 219 EGF-4 DLL3-14 bispecific
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGLGL- EWMGIINPSDGSTSYAQ
xF12Q molecule
KFQGRVTMTRDTSTNTVYMDLSSLRSEDTAVYYCARGGNSAFYSYYDMDVWGQGTTV- TVSSG
GGGSGGGGSGGGGSDVVMTQTPLSLPVTLGQPASISCRSSQSLVYRDGNTYLSWFQQRPGQS
PRRLIYKVSNWQSGVPDRFSGGGSGTDFTLKISRVEAEDVGVYYCMQGTHWPPTFGQGTKVE
IKSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFNSYAMNWVRQAPGKGLEWVARIRSK
YNNYATYYADSVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWWAYWG
QGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWV
QQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVF
GGGTKLTVL 220 EGF-4 DLL3-15 bispecific
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYIHWVRQAPGQGL- EWMGIINPSDGSTSYGQ
xF12Q molecule
NFQGRVTMTRDTSTNTVYMELSSLRSEDTAVYYCARGGNSAFYSYYDMDVWGQGTTV- TVSSG
GGGSGGGGSGGGGSDVVMTQSPLSLPVTLGQPASISCRSSQSLAYRDGNTYLSWFQQRPGQS
PRRLIYKVSNWQSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCMQGTHWPPTFGQGTKVE
IKSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFNSYAMNWVRQAPGKGLEWVARIRSK
YNNYATYYADSVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWWAYWG
QGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWV
QQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVF
GGGTKLTVL 221 N-term. DLL3-1 bispecific
QVQLVESGGGVVQSGRSLRLSCAASGFTFSDYGIHWVRQAPGKG- LEWVAVISYHGSNKYYAR
xI2C- molecule -
SVKGRFTVSRDNSKNTLYLQMNSLRAEDTAVYYCAREIPFGMDVWGQGTTVTVSSGGGGSGG HALB
HALB
GGSGGGGSDIVMTQTPLSLPVTPGEPASISCRSSQSLLHSDGYNYLDWYLQKPGQSPQLLIY-
variant 1 variant 1
LGSNRASGVPDRFSGSGSGTDFTLTISRVEAEDVGVYYCMQALQTPLTFGGGTKVDIKSGGG
GSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYAT
YYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVT
VSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQ
APRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKL
TVLPGGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTC
VADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRL
VRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAAC
LLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLT
KVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPAD
LPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAA
ADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVE
VSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPC
FSALEVDETYVPKEFNAGTFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAAMD
DFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLHHHHHH 222 N-term. DLL3-2
bispecific QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQG-
LEWMGWINPNSGDTNYAQ xI2C- molecule -
KFQGRVTMTRDTSISTAYMELSRLTSDDTAVYYCARDANIAALDAFEIWGQGTMVTVSSGGG HALB
HALB
GSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYA-
variant 1 variant 1
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIKSGGGG
SEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATY
YADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTV
SSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQA
PRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLT
VLPGGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCV
ADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLV
RPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACL
LPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTK
VHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADL
PSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAA
DPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEV
SRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCF
SALEVDETYVPKEFNAGTFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAAMDD
FAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLHHHHHH 223 EGF-1 DLL3-3
bispecific QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLE-
WVAVISYHGRDTYYAR xI2C- molecule -
SVKGRFTISRDNSKNTLYLHMNSLRAEDTAVYYCARDGATVTSYYYSGMDVWGQGTTVTVSS HALB
HALB
GGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWFQQKPGKAPKSL-
IYLASSLQSGVPSKFSGSGSGTDFTLTISSLQPEDFATYYCQQYNFYPFTFGPGTKVDIKSG
GGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNY
ATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTL
VTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKP
GQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGT
KLTVLPGGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAK
TCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLP
RLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKA
ACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTD
LTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMP
ADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCC
AAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTL
VEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRR
PCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAV
MDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLHHHHHH 224 EGF-3 DLL3-4
bispecific QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLE-
WIGYVYYSGTTNYNPS xI2C- molecule -
LKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASIAVTGFYFDYWGQGTLVTVSSGGGGSG HALB
HALB
GGGSGGGGSEIVLTQSPGTLSLSPGERVTLSCRASQRVNNNYLAWYQQRPGQAPRLLIYGAS-
SRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDRSPLTFGGGTKLEIKSGGGGSE
VQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYA
DSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSS
GGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPR
GLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL
PGGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVAD
ESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRP
EVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLP
KLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVH
TECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPS
LAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADP
HECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSR
NLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSA
LEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFA
AFVEKCCKADDKETCFAEEGKKLVAASQAALGLHHHHHH 225 EGF-3 DLL3-5 bispecific
QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLE- WIGYIYYSGRTNYYPS
xI2C- molecule -
LKSRVTISIDTSKNQFSLKLSSVTAADTAVYYCARIAVAGFFFDYWGQGTLVTVSSGGGGSG HALB
HALB
GGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVNKNYLAWYQQKPGQAPRLLIYGAS-
SRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDRSPLTFGGGTKLEIKSGGGGSE
VQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYA
DSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSS
GGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPR
GLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL
PGGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVAD
ESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRP
EVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLP
KLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVH
TECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPS
LAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADP
HECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSR
NLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSA
LEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFA
AFVEKCCKADDKETCFAEEGKKLVAASQAALGLHHHHHH 226 EGF-3 DLL3-6 bispecific
QVQLQESGPGLVKPSETLSLTCTVSGASISSFYWSWIRQPPGKGLE- WIGYIYYSGTTNYNPS
xI2C- molecule -
LKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARIAVAGFFFDYWGQGTLVTVSSGGGGSG HALB
HALB
GGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVNKNYLAWYQQKPGQAPRLLIYGAS-
SRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDRSPLTFGGGTKVEIKSGGGGSE
VQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYA
DSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSS
GGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPR
GLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL
PGGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVAD
ESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRP
EVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLP
KLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVH
TECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPS
LAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADP
HECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSR
NLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSA
LEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFA
AFVEKCCKADDKETCFAEEGKKLVAASQAALGLHHHHHH 227 EGF-3 DLL3-7 bispecific
QVQLQESGPGLVKPSQTLSLTCTVSGASISSFYWSWIRQPPGKGLE- WIGYIYYSGTTNYNPS
xI2C- molecule -
LKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARIAVAGFFFDYWGQGTLVTVSSGGGGSG HALB
HALB
GGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVNKNYLAWYQQKPGQAPRLLIYGAS-
SRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDRSPLTFGGGTKVEIKSGGGGSE
VQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYA
DSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSS
GGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPR
GLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL
PGGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVAD
ESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRP
EVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLP
KLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVH
TECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPS
LAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADP
HECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSR
NLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSA
LEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFA
AFVEKCCKADDKETCFAEEGKKLVAASQAALGLHHHHHH 228 EGF-3 DLL3-8 bispecific
QVQLQEWGPGLVKPSETLSLTCTVSGASISSFYWSWIRQPPGKGLE- WIGYIYYSGTTNYNPS
xI2C- molecule -
LKSRVTISVDTSKNQLSLKLSSVTAADTAVYYCARIAVAGFFFDYWGQGTLVTVSSGGGGSG HALB
HALB
GGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVNKNYLAWYQQKPGQAPRLLIYGAS-
SRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDRSPLTFGGGTKVDIKSGGGGSE
VQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYA
DSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSS
GGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPR
GLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL
PGGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVAD
ESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRP
EVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLP
KLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVH
TECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPS
LAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADP
HECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSR
NLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSA
LEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFA
AFVEKCCKADDKETCFAEEGKKLVAASQAALGLHHHHHH 229 EGF-3 DLL3-9 bispecific
QVQLQESGPGLVKPSETLSLTCTVSGASISSFYWSWIRQPPGKGLE- WIGYIYYSGTTNYNPS
xI2C- molecule -
LKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARIAVAGFFFDYWGQGTLVTVSSGGGGSG HALB
HALB
GGGSGGGGSEIVLTQSPGTLSLSPGESATLSCRASQSVNKNYLAWYQQKPGQAPRLLIYGAS-
SRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDRSPLTFGGGTRLEIKSGGGGSE
VQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYA
DSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSS
GGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPR
GLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL
PGGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVAD
ESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRP
EVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLP
KLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVH
TECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPS
LAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADP
HECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSR
NLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSA
LEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFA
AFVEKCCKADDKETCFAEEGKKLVAASQAALGLHHHHHH 230 EGF-3 DLL3-10
bispecific QVQLQESGPGLVKPSQTLSLTCTVSGGSISSYYWSWIRQPPGKGL-
EWIGYIFYNGITNYNPS xI2C- molecule -
LKSRVTISLDTSKNQFSLKLSSVTAADTAKYYCARIHSGSFSFDYWDQGTLVTVSSGGGGSG HALB
HALB
GGGSGGGGSEIVMTQSPGTLSLSPGERATLSCRASQSVSRGYLAWYQQKPGQAPRLLIYGAS-
SRATDIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDTSPITFGQGTKVEIKSGGGGSE
VQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYA
DSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSS
GGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPR
GLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL
PGGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVAD
ESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRP
EVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLP
KLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVH
TECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPS
LAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADP
HECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSR
NLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSA
LEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFA
AFVEKCCKADDKETCFAEEGKKLVAASQAALGLHHHHHH 231 EGF-3 DLL3-11
bispecific EVQLVESGGGLVKPGGSLRLSCAASGFIFNNAGMSWVRQAPGKGL-
EWVGRIKNKIDGGTTDF xI2C- molecule -
AAPVKGRFTISRDDSKNTLYLQMNSLKAEDTAVYYCTARGWYGDYFDYWGQGTLVTVSSGGG HALB
HALB
GSGGGGSGGGGSDIVMTQTPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQ-
LLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCMQALQTPFTFGPGTKVEIK
SGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYN
NYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQG
TLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ
KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGG
GTKLTVLPGGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEF
AKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPN
LPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAAD
KAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLV
TDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDE
MPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEK
CCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTP
TLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVN
RRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLK
AVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLHHHHHH 232 EGF-3/[4]
DLL3-12 bispecific QVQLVESGGGVVQPGRSLRLSCAASGFSFSSYDIHWVRQAP-
GKGLEWVAVISSHGSNKNYAR xI2C- molecule -
SVKGRFTISRDNSKNTLYLQMNSLKAEDTAVYYCARDGYSGNDPFYYYYHGMDVWGQGTTVT HALB
HALB
VSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAP-
KLLIYAASSLQSGVPSRFSGSGSGTDFSLTISSLQPEDFATYYCQQSFTTPLTFGGGTKVEI
KSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKY
NNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQ
GTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQ
QKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFG
GGTKLTVLPGGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTE
FAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNP
NLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAA
DKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKL
VTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVEND
EMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLE
KCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVST
PTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLV
NRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQL
KAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLHHHHHH 233 EGF-4 DLL3-13
bispecific QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGL-
EWMGIINPSDGSTNYAQ xI2C- molecule -
NFQGRVTMTRDTSTNTVYMELSSLRSEDTAVYYCARGGNSAFYSYYDMDVWGQGTTVTVSSG HALB
HALB
GGGSGGGGSGGGGSDVVMTQSPLSLPVTLGQPASISCRSSQSLVYRDGNTYLSWFQQRPGQS-
PRRLIYKVSNWQSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCMQGTHWPPTFGQGTKVE
IKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSK
YNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWG
QGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWV
QQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVF
GGGTKLTVLPGGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVT
EFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDN
PNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQA
ADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSK
LVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVEN
DEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTL
EKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVS
TPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESL
VNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQ
LKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLHHHHHH 234 EGF-4 DLL3-14
bispecific QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGLGL-
EWMGIINPSDGSTSYAQ xI2C- molecule -
KFQGRVTMTRDTSTNTVYMDLSSLRSEDTAVYYCARGGNSAFYSYYDMDVWGQGTTVTVSSG HALB
HALB
GGGSGGGGSGGGGSDVVMTQTPLSLPVTLGQPASISCRSSQSLVYRDGNTYLSWFQQRPGQS-
PRRLIYKVSNWQSGVPDRFSGGGSGTDFTLKISRVEAEDVGVYYCMQGTHWPPTFGQGTKVE
IKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSK
YNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWG
QGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWV
QQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVF
GGGTKLTVLPGGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVT
EFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDN
PNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQA
ADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSK
LVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVEN
DEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTL
EKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVS
TPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESL
VNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQ
LKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLHHHHHH 235 EGF-4 DLL3-15
bispecific QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYIHWVRQAPGQGL-
EWMGIINPSDGSTSYGQ xI2C- molecule -
NFQGRVTMTRDTSTNTVYMELSSLRSEDTAVYYCARGGNSAFYSYYDMDVWGQGTTVTVSSG HALB
HALB
GGGSGGGGSGGGGSDVVMTQSPLSLPVTLGQPASISCRSSQSLAYRDGNTYLSWFQQRPGQS-
PRRLIYKVSNWQSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCMQGTHWPPTFGQGTKVE
IKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSK
YNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWG
QGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWV
QQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVF
GGGTKLTVLPGGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVT
EFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDN
PNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQA
ADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSK
LVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVEN
DEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTL
EKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVS
TPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESL
VNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQ
LKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLHHHHHH 236 EGF-4 DLL3-16
bispecific QVQLVQSGAEVKKPGASVKVSCKASGYTFTGHYMHWVRQAPGQGL-
EWMGIINPSDGSTNYAQ xI2C- molecule -
KFQGRVTMTRDTSTSTVYMELRSLRSEDTAVYYCTRGTTVVHYSYYDMDVWGQGTTVTVSSG HALB
HALB
GGGSGGGGSGGGGSDVVMTQTPLSLPVTLGQPASISCRSSQSLVYRDGNTYLTWFQQRPGQS-
PRRLIYKVSNWQSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPPTFGGGTKVE
IKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSK
YNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWG
QGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWV
QQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVF
GGGTKLTVLPGGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVT
EFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDN
PNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQA
ADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSK
LVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVEN
DEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTL
EKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVS
TPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESL
VNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQ
LKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLHHHHHH 237 EGF-4 DLL3-17
bispecific QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYFMHWVRQAPGLGL-
EWMGIINPSDGSTSYAQ xI2C- molecule -
NFQGRVTMTRDTSTNTVYMELSSLRSEDTAVYYCARGGNSAFYSYYDMDVWGQGTTVTVSSG HALB
HALB
GGGSGGGGSGGGGSDVVMTQSPLSLPVTLGQPASISCRSSQSLVYRDGNTYLSWFQQRPGQS-
PRRLIYRVSNWQSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCMQGTYWPPTFGQGTKVD
IKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSK
YNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWG
QGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWV
QQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVF
GGGTKLTVLPGGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVT
EFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDN
PNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQA
ADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSK
LVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVEN
DEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTL
EKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVS
TPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESL
VNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQ
LKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLHHHHHH 238 EGF-5/[6]
DLL3-18 bispecific QVQLVESGGGAVQPGRSLRLSCAASGFTFSNYGMHWVRQAP-
GKGLEWVAVISHHGSSKYYAR xI2C- molecule -
SVKGRFTISRDNSKNTLYLEMNSLRAEDTAVYYCARDWWELVFDYWGQGTLVTVSSGGGGSG HALB
HALB
GGGSGGGGSDIVMTQTPLSLSVTPGQPASISCKSSQSLLHSDGKTFLYWYLQKPGQPPQLLI-
YEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQGIHLPFTFGPGTKVEIKSGG
GGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYA
TYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLV
TVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPG
QAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTK
LTVLPGGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKT
CVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPR
LVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAA
CLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDL
TKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPA
DLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCA
AADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLV
EVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRP
CFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVM
DDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLHHHHHH 239 EGF-5/[6] DLL3-19
bispecific QVTLKESGPMLVKPTETLTLTCTVSGFSLSNSRMGVSWIRQ-
PPGRALEWLAHIFSNDGKSYS xI2C- molecule -
TSLKSRLTISKDTSKSQVVLTMTNMDPVDTATYYCARYNYDSSGYYYSFFDYWGQGTLVTVS HALB
HALB
SGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKL-
LIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYSSPFTFGGGTKVEIKS
GGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNN
YATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGT
LVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQK
PGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGG
TKLTVLPGGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFA
KTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNL
PRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADK
AACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVT
DLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEM
PADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKC
CAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPT
LVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNR
RPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKA
VMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLHHHHHH 240 EGF-5/[6] DLL3-20
bispecific QVTLKESGPVLVKPTETLTLTCTVSGFSLSNARMGVSWLRQ-
PPGKALEWLAHIFSTDEKSYS xI2C- molecule -
TSLKSRLTISKDTSKSQVVLTMTNMDPVDTATYYCARYYYDSSGYYYSFFDYWGQGTLVTVS HALB
HALB
SGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSIRSYLNWYQQKPGKAPKL-
LIYGASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSSPFTFGGGTKVEIKS
GGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNN
YATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGT
LVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQK
PGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGG
TKLTVLPGGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFA
KTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNL
PRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADK
AACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVT
DLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEM
PADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKC
CAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPT
LVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNR
RPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKA
VMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLHHHHHH 241 EGF-5/[6] DLL3-21
bispecific QVQLVQSGAEVKKPGASVKVSCKASGYAFTSYYIHWVRQAP-
GQGLEWMGIINPSGGSKSYAQ xI2C- molecule -
KFRGRVTMTRDTSTSTVYMELSSLTSEDTAVYYCARSMSTVTSDAFDIWGQGTMVTVSSGGG HALB
HALB
GSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQKPGKAPKLLIYA-
ASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQSYSAPLTFGGGTKVDIKSGGGG
SEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATY
YADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTV
SSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQA
PRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLT
VLPGGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCV
ADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLV
RPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACL
LPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTK
VHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADL
PSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAA
DPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEV
SRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCF
SALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDD
FAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLHHHHHH 242 EGF-3 DLL3-4
bispecific QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLE-
WIGYVYYSGTTNYNPS xF12Q- molecule -
LKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASIAVTGFYFDYWGQGTLVTVSSGGGGSG HALB
HALB
GGGSGGGGSEIVLTQSPGTLSLSPGERVTLSCRASQRVNNNYLAWYQQRPGQAPRLLIYGAS-
SRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDRSPLTFGGGTKLEIKSGGGGSE
VQLVESGGGLVQPGGSLRLSCAASGFTFNSYAMNWVRQAPGKGLEWVARIRSKYNNYATYYA
DSVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWWAYWGQGTLVTVSS
GGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPR
GLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL
PGGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVAD
ESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRP
EVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLP
KLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVH
TECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPS
LAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADP
HECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSR
NLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSA
LEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFA
AFVEKCCKADDKETCFAEEGKKLVAASQAALGLHHHHHH 243 EGF-3 DLL3-5 bispecific
QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLE- WIGYIYYSGRTNYYPS
xF12Q- molecule -
LKSRVTISIDTSKNQFSLKLSSVTAADTAVYYCARIAVAGFFFDYWGQGTLVTVSSGGGGSG HALB
HALB
GGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVNKNYLAWYQQKPGQAPRLLIYGAS-
SRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDRSPLTFGGGTKLEIKSGGGGSE
VQLVESGGGLVQPGGSLRLSCAASGFTFNSYAMNWVRQAPGKGLEWVARIRSKYNNYATYYA
DSVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWWAYWGQGTLVTVSS
GGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPR
GLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL
PGGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVAD
ESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRP
EVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLP
KLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVH
TECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPS
LAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADP
HECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSR
NLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSA
LEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFA
AFVEKCCKADDKETCFAEEGKKLVAASQAALGLHHHHHH 244 EGF-3 DLL3-6 bispecific
QVQLQESGPGLVKPSETLSLTCTVSGASISSFYWSWIRQPPGKGLE- WIGYIYYSGTTNYNPS
xF12Q- molecule -
LKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARIAVAGFFFDYWGQGTLVTVSSGGGGSG HALB
HALB
GGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVNKNYLAWYQQKPGQAPRLLIYGAS-
SRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDRSPLTFGGGTKVEIKSGGGGSE
VQLVESGGGLVQPGGSLRLSCAASGFTFNSYAMNWVRQAPGKGLEWVARIRSKYNNYATYYA
DSVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWWAYWGQGTLVTVSS
GGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPR
GLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL
PGGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVAD
ESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRP
EVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLP
KLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVH
TECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPS
LAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADP
HECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSR
NLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSA
LEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFA
AFVEKCCKADDKETCFAEEGKKLVAASQAALGLHHHHHH 245 EGF-3 DLL3-7 bispecific
QVQLQESGPGLVKPSQTLSLTCTVSGASISSFYWSWIRQPPGKGLE- WIGYIYYSGTTNYNPS
xF12Q- molecule -
LKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARIAVAGFFFDYWGQGTLVTVSSGGGGSG HALB
HALB
GGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVNKNYLAWYQQKPGQAPRLLIYGAS-
SRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDRSPLTFGGGTKVEIKSGGGGSE
VQLVESGGGLVQPGGSLRLSCAASGFTFNSYAMNWVRQAPGKGLEWVARIRSKYNNYATYYA
DSVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWWAYWGQGTLVTVSS
GGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPR
GLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL
PGGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVAD
ESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRP
EVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLP
KLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVH
TECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPS
LAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADP
HECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSR
NLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSA
LEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFA
AFVEKCCKADDKETCFAEEGKKLVAASQAALGLHHHHHH 246 EGF-3 DLL3-8 bispecific
QVQLQEWGPGLVKPSETLSLTCTVSGASISSFYWSWIRQPPGKGLE- WIGYIYYSGTTNYNPS
xF12Q- molecule -
LKSRVTISVDTSKNQLSLKLSSVTAADTAVYYCARIAVAGFFFDYWGQGTLVTVSSGGGGSG HALB
HALB
GGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVNKNYLAWYQQKPGQAPRLLIYGAS-
SRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDRSPLTFGGGTKVDIKSGGGGSE
VQLVESGGGLVQPGGSLRLSCAASGFTFNSYAMNWVRQAPGKGLEWVARIRSKYNNYATYYA
DSVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWWAYWGQGTLVTVSS
GGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPR
GLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL
PGGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVAD
ESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRP
EVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLP
KLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVH
TECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPS
LAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADP
HECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSR
NLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSA
LEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFA
AFVEKCCKADDKETCFAEEGKKLVAASQAALGLHHHHHH 247 EGF-3 DLL3-9 bispecific
QVQLQESGPGLVKPSETLSLTCTVSGASISSFYWSWIRQPPGKGLE- WIGYIYYSGTTNYNPS
xF12Q- molecule -
LKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARIAVAGFFFDYWGQGTLVTVSSGGGGSG HALB
HALB
GGGSGGGGSEIVLTQSPGTLSLSPGESATLSCRASQSVNKNYLAWYQQKPGQAPRLLIYGAS-
SRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDRSPLTFGGGTRLEIKSGGGGSE
VQLVESGGGLVQPGGSLRLSCAASGFTFNSYAMNWVRQAPGKGLEWVARIRSKYNNYATYYA
DSVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWWAYWGQGTLVTVSS
GGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPR
GLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL
PGGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVAD
ESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRP
EVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLP
KLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVH
TECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPS
LAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADP
HECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSR
NLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSA
LEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFA
AFVEKCCKADDKETCFAEEGKKLVAASQAALGLHHHHHH 248 EGF-3 DLL3-10
bispecific QVQLQESGPGLVKPSQTLSLTCTVSGGSISSYYWSWIRQPPGKGL-
EWIGYIFYNGITNYNPS xF12Q- molecule -
LKSRVTISLDTSKNQFSLKLSSVTAADTAKYYCARIHSGSFSFDYWDQGTLVTVSSGGGGSG HALB
HALB
GGGSGGGGSEIVMTQSPGTLSLSPGERATLSCRASQSVSRGYLAWYQQKPGQAPRLLIYGAS-
SRATDIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDTSPITFGQGTKVEIKSGGGGSE
VQLVESGGGLVQPGGSLRLSCAASGFTFNSYAMNWVRQAPGKGLEWVARIRSKYNNYATYYA
DSVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWWAYWGQGTLVTVSS
GGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPR
GLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL
PGGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVAD
ESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRP
EVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLP
KLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVH
TECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPS
LAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADP
HECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSR
NLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSA
LEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFA
AFVEKCCKADDKETCFAEEGKKLVAASQAALGLHHHHHH 249 EGF-4 DLL3-13
bispecific QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGL-
EWMGIINPSDGSTNYAQ xF12Q- molecule -
NFQGRVTMTRDTSTNTVYMELSSLRSEDTAVYYCARGGNSAFYSYYDMDVWGQGTTVTVSSG HALB
HALB
GGGSGGGGSGGGGSDVVMTQSPLSLPVTLGQPASISCRSSQSLVYRDGNTYLSWFQQRPGQS-
PRRLIYKVSNWQSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCMQGTHWPPTFGQGTKVE
IKSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFNSYAMNWVRQAPGKGLEWVARIRSK
YNNYATYYADSVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWWAYWG
QGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWV
QQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVF
GGGTKLTVLPGGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVT
EFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDN
PNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQA
ADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSK
LVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVEN
DEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTL
EKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVS
TPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESL
VNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQ
LKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLHHHHHH 250 EGF-4 DLL3-14
bispecific QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGLGL-
EWMGIINPSDGSTSYAQ xF12Q- molecule -
KFQGRVTMTRDTSTNTVYMDLSSLRSEDTAVYYCARGGNSAFYSYYDMDVWGQGTTVTVSSG HALB
HALB
GGGSGGGGSGGGGSDVVMTQTPLSLPVTLGQPASISCRSSQSLVYRDGNTYLSWFQQRPGQS-
PRRLIYKVSNWQSGVPDRFSGGGSGTDFTLKISRVEAEDVGVYYCMQGTHWPPTFGQGTKVE
IKSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFNSYAMNWVRQAPGKGLEWVARIRSK
YNNYATYYADSVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWWAYWG
QGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWV
QQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVF
GGGTKLTVLPGGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVT
EFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDN
PNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQA
ADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSK
LVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVEN
DEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTL
EKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVS
TPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESL
VNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQ
LKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLHHHHHH 251 EGF-4 DLL3-15
bispecific QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYIHWVRQAPGQGL-
EWMGIINPSDGSTSYGQ xF12Q- molecule -
NFQGRVTMTRDTSTNTVYMELSSLRSEDTAVYYCARGGNSAFYSYYDMDVWGQGTTVTVSSG HALB
HALB
GGGSGGGGSGGGGSDVVMTQSPLSLPVTLGQPASISCRSSQSLAYRDGNTYLSWFQQRPGQS-
PRRLIYKVSNWQSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCMQGTHWPPTFGQGTKVE
IKSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFNSYAMNWVRQAPGKGLEWVARIRSK
YNNYATYYADSVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWWAYWG
QGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWV
QQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVF
GGGTKLTVLPGGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVT
EFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDN
PNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQA
ADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSK
LVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVEN
DEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTL
EKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVS
TPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESL
VNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQ
LKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLHHHHHH 252 -- Human human
MVSPRMSGLLSQTVILALIFLPQTRPAGVFELQIHSFGPGPGPGAPRSPCSARLP- CRLFFRV
DLL3 CLKPGLSEEAAESPCALGAALSARGPVYTEQPGAPAPDLPLPDGLLQVPFRDAWPGTFSFII
ETWREELGDQIGGPAWSLLARVAGRRRLAAGGPWARDIQRAGAWELRFSYRARCEPPAVGTA
CTRLCRPRSAPSRCGPGLRPCAPLEDECEAPLVCRAGCSPEHGFCEQPGECRCLEGWTGPLC
TVPVSTSSCLSPRGPSSATTGCLVPGPGPCDGNPCANGGSCSETPRSFECTCPRGFYGLRCE
VSGVTCADGPCFNGGLCVGGADPDSAYICHCPPGFQGSNCEKRVDRCSLQPCRNGGLCLDLG
HALRCRCRAGFAGPRCEHDLDDCAGRACANGGTCVEGGGAHRCSCALGFGGRDCRERADPCA
ARPCAHGGRCYAHFSGLVCACAPGYMGARCEFPVHPDGASALPAAPPGLRPGDPQRYLLPPA
LGLLVAAGVAGAALLLVHVRRRGHSQDAGSRLLAGTPEPSVHALPDALNNLRTQEGSGDGPS
SSVDWNRPEDVDPQGIYVISAPSIYAREVATPLFPPLHTGRAGQRQHLLFPYPSSILSVK 253 --
Human human
MVSPRMSGLLSQTVILALIFLPQTRPAGVFELQIHSFGPGPGPGAPRSPCSARLP- CRLFFRV
DLL3 CLKPGLSEEAAESPCALGAALSARGPVYTEQPGAPAPDLPLPDGLLQVPFRDAWPGTFSFII
ECD ETWREELGDQIGGPAWSLLARVAGRRRLAAGGPWARDIQRAGAWELRFSYRARCEPPAVGTA
CTRLCRPRSAPSRCGPGLRPCAPLEDECEAPLVCRAGCSPEHGFCEQPGECRCLEGWTGPLC
TVPVSTSSCLSPRGPSSATTGCLVPGPGPCDGNPCANGGSCSETPRSFECTCPRGFYGLRCE
VSGVTCADGPCFNGGLCVGGADPDSAYICHCPPGFQGSNCEKRVDRCSLQPCRNGGLCLDLG
HALRCRCRAGFAGPRCEHDLDDCAGRACANGGTCVEGGGAHRCSCALGFGGRDCRERADPCA
ARPCAHGGRCYAHFSGLVCACAPGYMGARCEFPVHPDGASALPAAPPGLRPGDPQRYL 254 --
Hu DLL3 human
MVSPRMSGLLSQTVILALIFLPQTRPAGVFELQIHSFGPGPGPGAPRSPCSAR- LPCRLFFRV
N-term.
CLKPGLSEEAAESPCALGAALSARGPVYTEQPGAPAPDLPLPDGLLQVPFRDAWPGTFSFII
ETWREELGDQIGGPAWSLLARVAGRRRLAAGGPWARDIQRAGAWELRFSYR 255 -- Hu DLL3
human ARCEPPAVGTACTRLCRPRSAPSRCGPGLRPCAPLEDECE DSL dom 256 -- Hu
DLL3 human APLVCRAGCSPEHGFCEQPGECRCLEGWTGPLCT EGF-1 257 -- Hu DLL3
human GPGPCDGNPCANGGSCSETPRSFECTCPRGFYGLRCE EGF-2 258 -- Hu DLL3
human SGVTCADGPCFNGGLCVGGADPDSAYICHCPPGFQGSNCE EGF-3 259 -- Hu DLL3
human RVDRCSLQPCRNGGLCLDLGHALRCRCRAGFAGPRCE EGF-4 260 -- Hu DLL3
human SGVTCADGPCFNGGLCVGGADPDSAYICHCPPGFQGSNCEKRVDRCSLQPCRN-
GGLCLDLGH EGF-3 + 4 ALRCRCRAGFAGPRCE 261 -- Hu DLL3 human
DLDDCAGRACANGGTCVEGGGAHRCSCALGFGGRDCR EGF-5 262 -- Hu DLL3 human
RADPCAARPCAHGGRCYAHFSGLVCACAPGYMGARCE EGF-6 263 -- Human artificial
MVSPRMSGLLSQTVILALIFLPQTRPAGVFELQIHSFGPGPGPGAPRSPC- SARLPCRLFFRV
DLL3 CLKPGLSEEAAESPCALGAALSARGPVYTEQPGAPAPDLPLPDGLLQVPFRDAWPGTFSFII
ECDx ETWREELGDQIGGPAWSLLARVAGRRRLAAGGPWARDIQRAGAWELRFSYRARCEPPAVGTA
EpCAM
CTRLCRPRSAPSRCGPGLRPCAPLEDECEAPLVCRAGCSPEHGFCEQPGECRCLEGWTGPLC
TVPVSTSSCLSPRGPSSATTGCLVPGPGPCDGNPCANGGSCSETPRSFECTCPRGFYGLRCE
VSGVTCADGPCFNGGLCVGGADPDSAYICHCPPGFQGSNCEKRVDRCSLQPCRNGGLCLDLG
HALRCRCRAGFAGPRCEHDLDDCAGRACANGGTCVEGGGAHRCSCALGFGGRDCRERADPCA
ARPCAHGGRCYAHFSGLVCACAPGYMGARCEFPVHPDGASALPAAPPGLRPGDPQRYLSGGG
GSGAGVIAVIVVVVIAIVAGIVVLVISRKKRMAKYEKAEIKEMGEMHRELNA 264 -- V5 x hu
artificial
MGWSCIILFLVATATGVHSGKPIPNPLLGLDSTSGARCEPPAVGTACTRLCRPRSAPSR- CGP
DLL3-
GLRPCAPLEDECEAPLVCRAGCSPEHGFCEQPGECRCLEGWTGPLCTVPVSTSSCLSPRGPS DSL
x SATTGCLVPGPGPCDGNPCANGGSCSETPRSFECTCPRGFYGLRCEVSGVTCADGPCFNGGL
EpCAM
CVGGADPDSAYICHCPPGFQGSNCEKRVDRCSLQPCRNGGLCLDLGHALRCRCRAGFAGPRC
EHDLDDCAGRACANGGTCVEGGGAHRCSCALGFGGRDCRERADPCAARPCAHGGRCYAHFSG
LVCACAPGYMGARCEFPVHPDGASALPAAPPGLRPGDPQRYLSGGGGSGAGVIAVIVVVVIA
IVAGIVVLVISRKKRMAKYEKAEIKEMGEMHRELNA 265 -- V5 x hu artificial
MGWSCIILFLVATATGVHSGKPIPNPLLGLDSTSGAPLVCRAGCSPEHGFCEQPGECRC- LEG
DLL3-
WTGPLCTVPVSTSSCLSPRGPSSATTGCLVPGPGPCDGNPCANGGSCSETPRSFECTCPRGF EGF1
x YGLRCEVSGVTCADGPCFNGGLCVGGADPDSAYICHCPPGFQGSNCEKRVDRCSLQPCRNGG
EpCAM
LCLDLGHALRCRCRAGFAGPRCEHDLDDCAGRACANGGTCVEGGGAHRCSCALGFGGRDCRE
RADPCAARPCAHGGRCYAHFSGLVCACAPGYMGARCEFPVHPDGASALPAAPPGLRPGDPQR
YLSGGGGSGAGVIAVIVVVVIAIVAGIVVLVISRKKRMAKYEKAEIKEMGEMHRELNA 266 --
V5 x hu artificial
MGWSCIILFLVATATGVHSGKPIPNPLLGLDSTSGGPGPCDGNPCANGGSCSETPRSFE- CTC
DLL3-
PRGFYGLRCEVSGVTCADGPCFNGGLCVGGADPDSAYICHCPPGFQGSNCEKRVDRCSLQPC EGF2
x RNGGLCLDLGHALRCRCRAGFAGPRCEHDLDDCAGRACANGGTCVEGGGAHRCSCALGFGGR
EpCAM
DCRERADPCAARPCAHGGRCYAHFSGLVCACAPGYMGARCEFPVHPDGASALPAAPPGLRPG
DPQRYLSGGGGSGAGVIAVIVVVVIAIVAGIVVLVISRKKRMAKYEKAEIKEMGEMHRELNA 267
-- V5 x hu artificial
MGWSCIILFLVATATGVHSGKPIPNPLLGLDSTSGSGVTCADGPCFNGGLCVGGADPDS- AYI
DLL3 -
CHCPPGFQGSNCEKRVDRCSLQPCRNGGLCLDLGHALRCRCRAGFAGPRCEHDLDDCAGRAC EGF3
x ANGGTCVEGGGAHRCSCALGFGGRDCRERADPCAARPCAHGGRCYAHFSGLVCACAPGYMGA
EpCAM
RCEFPVHPDGASALPAAPPGLRPGDPQRYLSGGGGSGAGVIAVIVVVVIAIVAGIVVLVISR
KKRMAKYEKAEIKEMGEMHRELNA 268 -- V5 x hu artificial
MGWSCIILFLVATATGVHSGKPIPNPLLGLDSTSGRVDRCSLQPCRNGGLCLDLGHALR- CRC
DLL3-
RAGFAGPRCEHDLDDCAGRACANGGTCVEGGGAHRCSCALGFGGRDCRERADPCAARPCAHG EGF4
x GRCYAHFSGLVCACAPGYMGARCEFPVHPDGASALPAAPPGLRPGDPQRYLSGGGGSGAGVI
EpCAM AVIVVVVIAIVAGIVVLVISRKKRMAKYEKAEIKEMGEMHRELNA 269 -- V5 x hu
artificial
MGWSCIILFLVATATGVHSGKPIPNPLLGLDSTSGDLDDCAGRACANGGTCVEGGGAHR- CSC
DLL3-
ALGFGGRDCRERADPCAARPCAHGGRCYAHFSGLVCACAPGYMGARCEFPVHPDGASALPAA EGF5
x PPGLRPGDPQRYLSGGGGSGAGVIAVIVVVVIAIVAGIVVLVISRKKRMAKYEKAEIKEMGE
EpCAM MHRELNA 270 -- V5 x hu artificial
MGWSCIILFLVATATGVHSGKPIPNPLLGLDSTSGRADPCAARPCAHGGRCYAHFSGLV- CAC
DLL3-
APGYMGARCEFPVHPDGASALPAAPPGLRPGDPQRYLSGGGGSGAGVIAVIVVVVIAIVAGI EGF6
x VVLVISRKKRMAKYEKAEIKEMGEMHRELNA EpCAM 271 -- Macaque cyno
MVSPRMSRLLSQTVILALIFIPQARPAGVFELQIHSFGPGPGPGAPRSPCSARG- PCRLFFRV
DLL3 CLKPGLSEEAAESPCALGAALSARGPVYTEQPEAPAPDLPLPNGLLQVPFRDAWPGTFSLII
ETWREELGDQIGGPAWSLLARVTRRRRLAAGGPWARDIQRAGAWELRFSYRARCELPAVGTA
CTRLCRPRSAPSRCGPGLRPCAPLEDECEAPPVCRAGCSLEHGFCEQPGECRCLEGWTGPLC
MVPASTSSCLGLRGPSSATTGCLVPGPGPCDGNPCANGGSCSETPGSFECTCPRGFYGLRCE
VSGVTCADGPCFNGGLCVGGADPDSAYICHCPPGFQGSNCEKRVDRCSLQPCRNGGLCLDLG
HALRCRCRAGFAGPRCEHNLDDCAGRACANGGTCVEGGGAHRCSCALGFGGRDCRERADPCA
ARPCAHGGRCYAHFSGLVCACAPGYMGSRCEFPVHPDGVSALPAAPPGLRPGDPQRYLLPPA
LGLLVAAGVAGAALLLVHVRRRGHAQDAGSRLLAGTPEPSVHALPDALNNQRTQEGPGDVPS
SSVDWNRPEDVDSRGIYVISAPSIYAREA 272 -- Macaque cyno
MVSPRMSRLLSQTVILALIFIPQARPAGVFELQIHSFGPGPGPGAPRSPCSARG- PCRLFFRV
DLL3 CLKPGLSEEAAESPCALGAALSARGPVYTEQPEAPAPDLPLPNGLLQVPFRDAWPGTFSLII
ECD ETWREELGDQIGGPAWSLLARVTRRRRLAAGGPWARDIQRAGAWELRFSYRARCELPAVGTA
CTRLCRPRSAPSRCGPGLRPCAPLEDECEAPPVCRAGCSLEHGFCEQPGECRCLEGWTGPLC
MVPASTSSCLGLRGPSSATTGCLVPGPGPCDGNPCANGGSCSETPGSFECTCPRGFYGLRCE
VSGVTCADGPCFNGGLCVGGADPDSAYICHCPPGFQGSNCEKRVDRCSLQPCRNGGLCLDLG
HALRCRCRAGFAGPRCEHNLDDCAGRACANGGTCVEGGGAHRCSCALGFGGRDCRERADPCA
ARPCAHGGRCYAHFSGLVCACAPGYMGSRCEFPVHPDGVSALPAAPPGLRPGDPQRYL 273 --
Ma DLL3 cyno
MVSPRMSRLLSQTVILALIFIPQARPAGVFELQIHSFGPGPGPGAPRSPCSARG- PCRLFFRV
N-term.
CLKPGLSEEAAESPCALGAALSARGPVYTEQPEAPAPDLPLPNGLLQVPFRDAWPGTFSLII
ETWREELGDQIGGPAWSLLARVTRRRRLAAGGPWARDIQRAGAWELRFSYR 274 -- Ma DLL3
cyno ARCELPAVGTACTRLCRPRSAPSRCGPGLRPCAPLEDECE DSL dom. 275 -- Ma
DLL3 cyno APPVCRAGCSLEHGFCEQPGECRCLEGWTGPLCM EGF-1
276 -- Ma DLL3 cyno GPGPCDGNPCANGGSCSETPGSFECTCPRGFYGLRCE EGF-2 277
-- Ma DLL3 cyno SGVTCADGPCFNGGLCVGGADPDSAYICHCPPGFQGSNCE EGF-3 278
-- Ma DLL3 cyno RVDRCSLQPCRNGGLCLDLGHALRCRCRAGFAGPRCE EGF-4 279 --
Ma DLL3 cyno
SGVTCADGPCFNGGLCVGGADPDSAYICHCPPGFQGSNCEKRVDRCSLQPCRNG- GLCLDLGH
EGF-3 + 4 ALRCRCRAGFAGPRCE 280 -- Ma DLL3 cyno
NLDDCAGRACANGGTCVEGGGAHRCSCALGFGGRDCR EGF-5 281 -- Ma DLL3 cyno
RADPCAARPCAHGGRCYAHFSGLVCACAPGYMGSRCE EGF-6 282 -- Ma DLL3
artificial MVSPRMSRLLSQTVILALIFIPQARPAGVFELQIHSFGPGPGPGAPRS-
PCSARGPCRLFFRV ECD x
CLKPGLSEEAAESPCALGAALSARGPVYTEQPEAPAPDLPLPNGLLQVPFRDAWPGTFSLII
EpCAM
ETWREELGDQIGGPAWSLLARVTRRRRLAAGGPWARDIQRAGAWELRFSYRARCELPAVGTA
CTRLCRPRSAPSRCGPGLRPCAPLEDECEAPPVCRAGCSLEHGFCEQPGECRCLEGWTGPLC
MVPASTSSCLGLRGPSSATTGCLVPGPGPCDGNPCANGGSCSETPGSFECTCPRGFYGLRCE
VSGVTCADGPCFNGGLCVGGADPDSAYICHCPPGFQGSNCEKRVDRCSLQPCRNGGLCLDLG
HALRCRCRAGFAGPRCEHNLDDCAGRACANGGTCVEGGGAHRCSCALGFGGRDCRERADPCA
ARPCAHGGRCYAHFSGLVCACAPGYMGSRCEFPVHPDGVSALPAAPPGLRPGDPQRYLSGGG
GSGAGVIAVIVVVVIAIVAGIVVLVISRKKRMAKYEKAEIKEMGEMHRELNA 283 -- Human
human MGSRCALALAVLSALLCQVWSSGVFELKLQEFVNKKGLLGNRNCCRGGAGPPPCA-
CRTFFRV DLL1
CLKHYQASVSPEPPCTYGSAVTPVLGVDSFSLPDGGGADSAFSNPIRFPFGFTWPGTFSLII
EALHTDSPDDLATENPERLISRLATQRHLTVGEEWSQDLHSSGRTDLKYSYRFVCDEHYYGE
GCSVFCRPRDDAFGHFTCGERGEKVCNPGWKGPYCTEPICLPGCDEQHGFCDKPGECKCRVG
WQGRYCDECIRYPGCLHGTCQQPWQCNCQEGWGGLFCNQDLNYCTHHKPCKNGATCTNTGQG
SYTCSCRPGYTGATCELGIDECDPSPCKNGGSCTDLENSYSCTCPPGFYGKICELSAMTCAD
GPCFNGGRCSDSPDGGYSCRCPVGYSGFNCEKKIDYCSSSPCSNGAKCVDLGDAYLCRCQAG
FSGRHCDDNVDDCASSPCANGGTCRDGVNDFSCTCPPGYTGRNCSAPVSRCEHAPCHNGATC
HERGHRYVCECARGYGGPNCQFLLPELPPGPAVVDLTEKLEGQGGPFPWVAVCAGVILVLML
LLGCAAVVVCVRLRLQKHRPPADPCRGETETMNNLANCQREKDISVSIIGATQIKNTNKKAD
FHGDHSADKNGFKARYPAVDYNLVQDLKGDDTAVRDAHSKRDTKCQPQGSSGEEKGTPTTLR
GGEASERKRPDSGCSTSKDTKYQSVYVISEEKDECVIATEV 284 -- Human human
MAAASRSASGWALLLLVALWQQRAAGSGVFQLQLQEFINERGVLASGRPCEPGCR- TFFRVCL
DLL4 KHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPFNFTWPGTFSLIIEAWHA
PGDDLRPEALPPDALISKIAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSR
LCKKRNDHFGHYVCQPDGNLSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGR
LCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSNSGQRSYTC
TCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCF
NGGSCRERNQGANYACECPPNFTGSNCEKKVDRCTSNPCANGGQCLNRGPSRMCRCRPGFTG
TYCELHVSDCARNPCAHGGTCHDLENGLMCTCPAGFSGRRCEVRTSIDACASSPCFNRATCY
TDLSTDTFVCNCPYGFVGSRCEFPVGLPPSFPWVAVSLGVGLAVLLVLLGMVAVAVRQLRLR
RPDDGSREAMNNLSDFQKDNLIPAAQLKNTNQKKELEVDCGLDKSNCGKQQNHTLDYNLAPG
PLGRGTMPGKFPHSDKSLGEKAPLRLHSEKPECRISAICSPRDSMYQSVCLISEERNECVIA TEV
285 -- linker 1 artificial GGGG 286 -- linker 2 artificial GGGGS
287 -- linker 3 artificial GGGGQ 288 -- linker 4 artificial SGGGGS
289 -- linker 5 artificial PGGGGS 290 -- linker 6 artificial PGGDGS
291 -- linker 7 artificial GGGGSGGGS 292 -- linker 8 artificial
GGGGSGGGGS 293 linker 9 artificial GGGGSGGGGSGGGGS 294 -- Hexa-his
artificial HHHHHH 295 -- Ab156 artificial RDWDFDVFGGGTPVGG 296 --
linear artificial QRFVTGHFGGLXPANG FcRn BP 297 -- linear artificial
QRFVTGHFGGLYPANG FcRn BP- Y 298 -- linear artificial
QRFVTGHFGGLHPANG FcRn BP- H 299 core FcRn artificial TGHFGGLHP BP-H
300 cyclic artificial QRFCTGHFGGLHPCNG FcRn BP- H 301 -- HALB human
DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVAD- ESAENC
DKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMC
TAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELR
DEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHG
DLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFV
ESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAK
VFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVG
SKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDET
YVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKC
CKADDKETCFAEEGKKLVAASQAALGL 302 -- HALB artificial
DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAK- TCVADESAENC
variant 1
DKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVM- C
TAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELR
DEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHG
DLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFV
ESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAK
VFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVG
SKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDET
YVPKEFNAGTFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAAMDDFAAFVEKC
CKADDKETCFAEEGKKLVAASQAALGL 303 -- HALB artificial
DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAK- TCVADESAENC
variant 2
DKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVM- C
TAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELR
DEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHG
DLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFV
ESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAK
VFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVG
SKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDET
YVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKC
CKADDKETCFAEEGPKLVAASQAALGL 304 -- HALB artificial
DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAK- TCVADESAENC
variant 3
DKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVM- C
TAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELR
DEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHG
DLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFV
ESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAK
VFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVG
SKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALDVDET
YVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKC
CKADDKETCFAEEGPHLVAASKAALGL 305 -- HALB artificial
DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAK- TCVADESAENC
variant 4
DKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVM- C
TAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELR
DEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHG
DLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFV
ESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAK
VFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVG
SKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALGVDET
YVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDKFAAFVEKC
CKADDKETCFAEEGPKLVAASQAALGL 306 -- HALB artificial
DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAK- TCVADESAENC
variant 5
DKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVM- C
TAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELR
DEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHG
DLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFV
ESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAK
VFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVG
SKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALDVDET
YVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDKFAAFVEKC
CKADDKETCFAEEGPKLVAASQAALGL 307 -- HALB artificial
DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAK- TCVADESAENC
variant 6
DKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVM- C
TAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELR
DEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHG
DLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFV
ESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAK
VFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVG
SKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDET
YVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDKFAAFVEKC
CKADDKETCFAEEGPHLVAASQAALGL 308 -- HALB artificial
DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAK- TCVADESAENC
variant 7
DKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVM- C
TAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELR
DEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHG
DLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFV
ESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAK
VFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVG
SKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDET
YVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKC
CKADDKETCFAEEGPHLVAASKAALGL 309 -- HALB artificial
DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAK- TCVADESAENC
variant 8
DKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVM- C
TAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELR
DEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHG
DLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFV
ESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAK
VFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVG
SKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDET
YVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDKFAAFVEKC
CKADDKETCFAEEGPKLVAASKAALGL 310 -- HALB artificial
DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAK- TCVADESAENC
variant 9
DKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVM- C
TAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELR
DEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHG
DLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFV
ESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAK
VFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVG
SKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALDVDET
YVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKC
CKADDKETCFAEEGPKLVAASKAALGL 311 -- HALB artificial
DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAK- TCVADESAENC
variant 10
DKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDV- MC
TAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELR
DEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHG
DLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFV
ESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAK
VFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVG
SKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDET
YVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKC
CKADDKETCFAEEGKKLVAASQAALGL 312 -- HALB artificial
DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAK- TCVADESAENC
variant 11
DKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDV- MC
TAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELR
DEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHG
DLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFV
ESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAK
VFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVG
SKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDET
YVPKEFNAGTFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAAMDDFAAFVEKC
CKADDKETCFAEEGKKLVAASQAALGL 313 -- HALB artificial
DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAK- TCVADESAENC
variant 12
DKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDV- MC
TAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELR
DEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHG
DLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFV
ESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAK
VFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVG
SKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDET
YVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKC
CKADDKETCFAEEGPKLVAASQAALGL 314 -- HALB artificial
DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAK- TCVADESAENC
variant 13
DKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDV- MC
TAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELR
DEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHG
DLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFV
ESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAK
VFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVG
SKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALDVDET
YVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKC
CKADDKETCFAEEGPHLVAASKAALGL 315 -- HALB artificial
DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAK- TCVADESAENC
variant 14
DKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDV- MC
TAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELR
DEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHG
DLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFV
ESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAK
VFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVG
SKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALGVDET
YVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDKFAAFVEKC
CKADDKETCFAEEGPKLVAASQAALGL 316 -- HALB artificial
DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAK- TCVADESAENC
variant 15
DKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDV- MC
TAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELR
DEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHG
DLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFV
ESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAK
VFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVG
SKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALDVDET
YVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDKFAAFVEKC
CKADDKETCFAEEGPKLVAASQAALGL 317 -- HALB artificial
DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAK- TCVADESAENC
variant 16
DKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDV- MC
TAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELR
DEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHG
DLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFV
ESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAK
VFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVG
SKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDET
YVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDKFAAFVEKC
CKADDKETCFAEEGPHLVAASQAALGL 318 -- HALB artificial
DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAK- TCVADESAENC
variant 17
DKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDV- MC
TAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELR
DEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHG
DLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFV
ESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAK
VFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVG
SKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDET
YVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKC
CKADDKETCFAEEGPHLVAASKAALGL 319 -- HALB artificial
DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAK- TCVADESAENC
variant 18
DKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDV- MC
TAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELR
DEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHG
DLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFV
ESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAK
VFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVG
SKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDET
YVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDKFAAFVEKC
CKADDKETCFAEEGPKLVAASKAALGL 320 -- HALB artificial
DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAK- TCVADESAENC
variant 19
DKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDV- MC
TAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELR
DEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHG
DLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFV
ESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAK
VFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVG
SKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALDVDET
YVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKC
CKADDKETCFAEEGPKLVAASKAALGL 321 -- HALB artificial
DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNEVTEFAK- TCVADESAENC
variant 20
DKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDV- MC
TAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELR
DEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHG
DLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFV
ESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAK
VFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVG
SKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDET
YVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKC
CKADDKETCFAEEGKKLVAASQAALGL 322 -- HALB artificial
DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNEVTEFAK- TCVADESAENC
variant 21
DKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDV- MC
TAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELR
DEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHG
DLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFV
ESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAK
VFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVG
SKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDET
YVPKEFNAGTFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAAMDDFAAFVEKC
CKADDKETCFAEEGKKLVAASQAALGL 323 -- HALB artificial
DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNEVTEFAK- TCVADESAENC
variant 22
DKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDV- MC
TAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELR
DEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHG
DLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFV
ESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAK
VFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVG
SKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDET
YVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKC
CKADDKETCFAEEGPKLVAASQAALGL 324 -- HALB artificial
DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNEVTEFAK- TCVADESAENC
variant 23
DKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDV- MC
TAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELR
DEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHG
DLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFV
ESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAK
VFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVG
SKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALDVDET
YVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKC
CKADDKETCFAEEGPHLVAASKAALGL 325 -- HALB artificial
DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNEVTEFAK- TCVADESAENC
variant 24
DKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDV- MC
TAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELR
DEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHG
DLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFV
ESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAK
VFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVG
SKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALGVDET
YVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDKFAAFVEKC
CKADDKETCFAEEGPKLVAASQAALGL 326 -- HALB artificial
DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNEVTEFAK- TCVADESAENC
variant 25
DKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDV- MC
TAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELR
DEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHG
DLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFV
ESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAK
VFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVG
SKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALDVDET
YVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDKFAAFVEKC
CKADDKETCFAEEGPKLVAASQAALGL 327 -- HALB artificial
DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNEVTEFAK- TCVADESAENC
variant 26
DKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDV- MC
TAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELR
DEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHG
DLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFV
ESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAK
VFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVG
SKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDET
YVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDKFAAFVEKC
CKADDKETCFAEEGPHLVAASQAALGL 328 -- HALB artificial
DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNEVTEFAK- TCVADESAENC
variant 27
DKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDV- MC
TAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELR
DEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHG
DLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFV
ESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAK
VFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVG
SKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDET
YVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKC
CKADDKETCFAEEGPHLVAASKAALGL 329 -- HALB artificial
DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNEVTEFAK- TCVADESAENC
variant 28
DKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDV- MC
TAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELR
DEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHG
DLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFV
ESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAK
VFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVG
SKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDET
YVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDKFAAFVEKC
CKADDKETCFAEEGPKLVAASKAALGL 330 -- HALB artificial
DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNEVTEFAK- TCVADESAENC
variant 29
DKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDV- MC
TAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELR
DEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHG
DLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFV
ESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAK
VFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVG
SKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALDVDET
YVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKC
CKADDKETCFAEEGPKLVAASKAALGL 331 -- Cross artificial
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV- HTFPAVLQSSGL
body 1
YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVF HC
LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCV
SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSL
TCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSV
MHEALHNHYTQKSLSLSPGK 332 -- Cross artificial
GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVK- AGVETTTPSKQS
body 1
NNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSDKTHTCPPCPAPELLGGP LC
SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQYGSTY
RCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKNQ
VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFS
CSVMHEALHNHYTQKSLSLSPGK 333 -- Cross artificial
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV- HTFPAVLQSSGL
body 2
YSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVEPKSSDKTHTCPPCPAPEAAGGPSVF HC
LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSL
TCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSV
MHEALHNHYTQKSLSLSPGK 334 -- Cross artificial
GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVK- AGVETTTPSKQS
body 2
NNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSEPKSSDKTHTCPPCPAPE LC
AAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ
YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKE
MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQ
GNVFSCSVMHEALHNHYTQKSLSLSPGK 335 -- Hetero-Fc artificial
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV- SHEDPEVKFNWYVDGV
binder Fc
EVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP- R
EPQVYTLPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFL
YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 336 -- Hetero-Fc
artificial DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV-
SHEDPEVKFNWYVDGV partner
EVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR Fc
EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFL
YSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 337 -- Maxi- artificial
EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD- VSHEDPEVKFNW
body 1
YVDGVEVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA
target Fc
KGQPREPQVYTLPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKS- D
GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 338 -- Maxi-
artificial EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD-
VSHEDPEVKFNW body 1
YVDGVEVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA CD3
Fc KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSD
GSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 339 -- Maxi-
artificial EPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVD-
VSHEDPEVKFNW body 2
YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA
target Fc
KGQPREPQVYTLPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKS- D
GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 340 -- Maxi-
artificial EPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVD-
VSHEDPEVKFNW body 2
YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA CD3
Fc KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSD
GSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 341 -- Mono
artificial
APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP- R Fc
EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVTTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSR
WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 342 -- CDR-L1 artificial
GSSTGAVTSGYYPN of F6A 343 CDR-L2 artificial GTKFLAP of F6A 344
CDR-L3 artificial ALWYSNRWV of F6A 345 CDR-H1 artificial IYAMN of
F6A 346 CDR-H2 artificial RIRSKYNNYATYYADSVKS of F6A 347 CDR-H3
artificial HGNFGNSYVSFFAY of F6A 348 VH of artificial
EVQLVESGGGLVQPGGSLKLSCAASGFTFNIYAMNWVRQAPGKGLEWVARIR- SKYNNYATYY
F6A ADSVKSRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSFFAYWGQGTLVTVS
S 349 VL of artificial
QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGG- TKFLAPGTPA
F6A RFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 350 VH-VL of
artificial EVQLVESGGGLVQPGGSLKLSCAASGFTFNIYAMNWVRQAPGKGLEWVA-
RIRSKYNNYATYY F6A
ADSVKSRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSFFAYWGQGTLVTVS
SGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAP
RGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTV L
351 CDR-L1 artificial GSSTGAVTSGYYPN of H2C 352 CDR-L2 artificial
GTKFLAP of H2C 353 CDR-L3 artificial ALWYSNRWV of H2C 354 CDR-H1
artificial KYAMN of H2C 355 CDR-H2 artificial RIRSKYNNYATYYADSVKD
of H2C 356 CDR-H3 artificial HGNFGNSYISYWAY of H2C 357 VH of
artificial EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIR-
SKYNNYATYY H2C
ADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVS S
358 VL of artificial
QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGG- TKFLAPGTPA
H2C RFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 359 VH-VL of
artificial EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVA-
RIRSKYNNYATYY H2C
ADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVS
SGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAP
RGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTV L
360 CDR-L1 artificial GSSTGAVTSGYYPN of H1E 361 CDR-L2 artificial
GTKFLAP of H1E 362 CDR-L3 artificial ALWYSNRWV of H1E 363 CDR-H1
artificial SYAMN of H1E 364 CDR-H2 artificial RIRSKYNNYATYYADSVKG
of H1E 365 CDR-H3 artificial HGNFGNSYLSFWAY of H1E 366 VH of
artificial EVQLVESGGGLEQPGGSLKLSCAASGFTFNSYAMNWVRQAPGKGLEWVARIR-
SKYNNYATYY H1E
ADSVKGRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYLSFWAYWGQGTLVTVS S
367 VL of artificial
QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGG- TKFLAPGTPA
H1E RFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 368 VH-VL of
artificial EVQLVESGGGLEQPGGSLKLSCAASGFTFNSYAMNWVRQAPGKGLEWVA-
RIRSKYNNYATYY H1E
ADSVKGRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYLSFWAYWGQGTLVTVS
SGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAP
RGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTV L
369 CDR-L1 artificial GSSTGAVTSGYYPN of G4H 370 CDR-L2 artificial
GTKFLAP of G4H 371 CDR-L3 artificial ALWYSNRWV of G4H 372 CDR-H1
artificial RYAMN of G4H 373 CDR-H2 artificial RIRSKYNNYATYYADSVKG
of G4H 374 CDR-H3 artificial HGNFGNSYLSYFAY of G4H 375 VH of
artificial EVQLVESGGGLVQPGGSLKLSCAASGFTFNRYAMNWVRQAPGKGLEWVARIR-
SKYNNYATYY G4H
ADSVKGRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYLSYFAYWGQGTLVTVS S
376 VL of artificial
QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGG- TKFLAPGTPA
G4H RFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 377 VH-VL of
artificial EVQLVESGGGLVQPGGSLKLSCAASGFTFNRYAMNWVRQAPGKGLEWVA-
RIRSKYNNYATYY G4H
ADSVKGRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYLSYFAYWGQGTLVTVS
SGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAP
RGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTV L
378 CDR-L1 artificial RSSTGAVTSGYYPN of A2J 379 CDR-L2 artificial
ATDMRPS of A2J 380 CDR-L3 artificial ALWYSNRWV of A2J 381 CDR-H1
artificial VYAMN of A2J 382 CDR-H2 artificial RIRSKYNNYATYYADSVKK
of A2J 383 CDR-H3 artificial HGNFGNSYLSWWAY of A2J 384 VH of A2J
artificial EVQLVESGGGLVQPGGSLKLSCAASGFTFNVYAMNWVRQAPGKGLEWV-
ARIRSKYNNYATYY
ADSVKKRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYLSWWAYWGQGTLVTVS S
385 VL of A2J artificial
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTSGYYPNWVQQKPGQAPRG- LIGATDMRPSGTPA
RFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 386 VH-VL of
artificial EVQLVESGGGLVQPGGSLKLSCAASGFTFNVYAMNWVRQAPGKGLEWVA-
RIRSKYNNYATYY A2J
ADSVKKRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYLSWWAYWGQGTLVTVS
SGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCRSSTGAVTSGYYPNWVQQKPGQAP
RGLIGATDMRPSGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTV L
387 CDR-L1 artificial GSSTGAVTSGYYPN of E1L 388 CDR-L2 artificial
GTKFLAP of E1L 389 CDR-L3 artificial ALWYSNRWV of E1L 390 CDR-H1
artificial KYAMN of E1L 391 CDR-H2 artificial RIRSKYNNYATYYADSVKS
of E1L 392 CDR-H3 artificial HGNFGNSYTSYYAY of E1L
393 VH of artificial
EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIR- SKYNNYATYY
E1L ADSVKSRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYTSYYAYWGQGTLVTVS
S 394 VL of E1L artificial
QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRG- LIGGTKFLAPGTPA
RFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 395 VH-VL of
artificial EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVA-
RIRSKYNNYATYY E1L
ADSVKSRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYTSYYAYWGQGTLVTVS
SGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAP
RGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTV L
396 CDR-L1 artificial RSSTGAVTSGYYPN of E2M 397 CDR-L2 artificial
ATDMRPS of E2M 398 CDR-L3 artificial ALWYSNRWV of E2M 399 CDR-H1
artificial GYAMN of E2M 400 CDR-H2 artificial RIRSKYNNYATYYADSVKE
of E2M 401 CDR-H3 artificial HRNFGNSYLSWFAY of E2M 402 VH of
artificial EVQLVESGGGLVQPGGSLKLSCAASGFTFNGYAMNWVRQAPGKGLEWVARIR-
SKYNNYATYY E2M
ADSVKERFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHRNFGNSYLSWFAYWGQGTLVTVS S
403 VL of artificial
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTSGYYPNWVQQKPGQAPRGLIGA- TDMRPSGTPA
E2M RFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 404 VH-VL of
artificial EVQLVESGGGLVQPGGSLKLSCAASGFTFNGYAMNWVRQAPGKGLEWVA-
RIRSKYNNYATYY E2M
ADSVKERFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHRNFGNSYLSWFAYWGQGTLVTVS
SGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCRSSTGAVTSGYYPNWVQQKPGQAP
RGLIGATDMRPSGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTV L
405 CDR-L1 artificial GSSTGAVTSGYYPN of F7O 406 CDR-L2 artificial
GTKFLAP of F7O 407 CDR-L3 artificial ALWYSNRWV of F7O 408 CDR-H1
artificial VYAMN of F7O 409 CDR-H2 artificial RIRSKYNNYATYYADSVKK
of F7O 410 CDR-H3 artificial HGNFGNSYISWWAY of F7O 411 VH of
artificial EVQLVESGGGLVQPGGSLKLSCAASGFTFNVYAMNWVRQAPGKGLEWVARIR-
SKYNNYATYY F7O
ADSVKKRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISWWAYWGQGTLVTVS S
412 VL of artificial
QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGG- TKFLAPGTPA
F7O RFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 413 VH-VL of
artificial EVQLVESGGGLVQPGGSLKLSCAASGFTFNVYAMNWVRQAPGKGLEWVA-
RIRSKYNNYATYY F7O
ADSVKKRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISWWAYWGQGTLVTVS
SGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAP
RGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTV L
414 CDR-L1 artificial GSSTGAVTSGNYPN of F12Q 415 CDR-L2 artificial
GTKFLAP of F12Q 416 CDR-L3 artificial VLWYSNRWV of F12Q 417 CDR-H1
artificial SYAMN of F12Q 418 CDR-H2 artificial RIRSKYNNYATYYADSVKG
of F12Q 419 CDR-H3 artificial HGNFGNSYVSWWAY of F12Q 420 VH of
artificial EVQLVESGGGLVQPGGSLKLSCAASGFTFNSYAMNWVRQAPGKGLEWVARIR-
SKYNNYATYY F12Q
ADSVKGRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWWAYWGQGTLVTVS S
421 VL of artificial
QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGG- TKFLAPGTPA
F12Q RFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 422 VH-VL of
artificial EVQLVESGGGLVQPGGSLKLSCAASGFTFNSYAMNWVRQAPGKGLEWVA-
RIRSKYNNYATYY F12Q
ADSVKGRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWWAYWGQGTLVTVS
SGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAP
RGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTV L
423 CDR-L1 artificial GSSTGAVTSGNYPN of I2C 424 CDR-L2 artificial
GTKFLAP of I2C 425 CDR-L3 artificial VLWYSNRWV of I2C 426 CDR-H1
artificial KYAMN of I2C 427 CDR-H2 artificial RIRSKYNNYATYYADSVKD
of I2C 428 CDR-H3 artificial HGNFGNSYISYWAY of I2C 429 VH of I2C
artificial EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWV-
ARIRSKYNNYATYY
ADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVS S
430 VL of I2C artificial
QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRG- LIGGTKFLAPGTPA
RFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 431 VH-VL of
artificial EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVA-
RIRSKYNNYATYY I2C
ADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVS
SGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAP
RGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTV L
432 VH of artificial
EVQLVESGGGLVQPGGSLRLSCAASGFTFNSYAMNWVRQAPGKGLEWVARIR- SKYNNYATYY
F12q ADSVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWWAYWGQGTLVTVS
S 433 VL of artificial
QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGG- TKFLAPGTPA
F12q RFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 434 VH-VL of
artificial EVQLVESGGGLVQPGGSLRLSCAASGFTFNSYAMNWVRQAPGKGLEWVA-
RIRSKYNNYATYY F12q
ADSVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWWAYWGQGTLVTVS
SGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAP
RGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTV L
435 DLL3-4- VH
QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKCLEWIGYVYYSGTTN- YNPS
001 LKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASIAVTGFYFDYWGQGTLVTVSS (G44C)
436 DLL3-4- VL
EIVLTQSPGTLSLSPGERVTLSCRASQRVNNNYLAWYQQRPGQAPRLLIYGASSRATG- IPDR
001 FSGSGSGTDFTLTISRLEPEDFAVYYCQQYDRSPLTFGCGTKLEIK (G234C) 437
DLL3-4- scFv
QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKCLEWIGYVYYSGT- TNYNPS
001 LKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASIAVTGFYFDYWGQGTLVTVSSGGGGSG
(G44C-
GGGSGGGGSEIVLTQSPGTLSLSPGERVTLSCRASQRVNNNYLAWYQQRPGQAPRLLIYGAS
G243C) SRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDRSPLTFGCGTKLEIK 438
DLL3-4- bispecific
QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKCLEWIGY- VYYSGTTNYNPS
001 (CC) molecule
LKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASIAVTGFYFDYWGQGTLVTV- SSGGGGSG
xI2C GGGSGGGGSEIVLTQSPGTLSLSPGERVTLSCRASQRVNNNYLAWYQQRPGQAPRLLIYGAS
SRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDRSPLTFGCGTKLEIKSGGGGSE
VQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYA
DSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSS
GGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPR
GLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 439
DLL3-14- VH-CDR2 IINPSEGSTSYAQKFQG D55E 440 DLL3-14- VH-CDR2
IINPSDASTSYAQKFQG G56A 441 DLL3-14- VL-CDR1 RSSQSLVYREGNTYLS D171E
442 DLL3-14- VL-CDR1 RSSQSLVYRDANTYLS G172A 443 DLL3-14- VL-CDR1
RSSQSLVYRDGQTYLS N173Q 444 DLL3-14- VL-CDR1 RSSQSLVYRDGNAYLS T174A
445 DLL3-14- VH
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGQGLEWMGIINPSDGS- TSYAQ
L43Q KFQGRVTMTRDTSTNTVYMDLSSLRSEDTAVYYCARGGNSAFYSYYDMDVWGQGTTVTVSS
446 DLL3-14- VH
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGLGLEWMGIINPSEGS- TSYAQ
D55E KFQGRVTMTRDTSTNTVYMDLSSLRSEDTAVYYCARGGNSAFYSYYDMDVWGQGTTVTVSS
447 DLL3-14- VH
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGLGLEWMGIINPSDAS- TSYAQ
G56A KFQGRVTMTRDTSTNTVYMDLSSLRSEDTAVYYCARGGNSAFYSYYDMDVWGQGTTVTVSS
448 DLL3-14- VH
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGQGLEWMGIINPSEGS- TSYAQ
L43Q- KFQGRVTMTRDTSTNTVYMDLSSLRSEDTAVYYCARGGNSAFYSYYDMDVWGQGTTVTVSS
D55E 449 DLL3-14- VH
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGQGLEWMGIINPSDAS- TSYAQ
L43Q- KFQGRVTMTRDTSTNTVYMDLSSLRSEDTAVYYCARGGNSAFYSYYDMDVWGQGTTVTVS
G56A 450 DLL3-14- VH
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGLCLEWMGIINPSDGS- TSYAQ
G44C KFQGRVTMTRDTSTNTVYMDLSSLRSEDTAVYYCARGGNSAFYSYYDMDVWGQGTTVTVSS
451 DLL3-14- VH
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGQCLEWMGIINPSDGS- TSYAQ
L43Q- KFQGRVTMTRDTSTNTVYMDLSSLRSEDTAVYYCARGGNSAFYSYYDMDVWGQGTTVTVSS
G44C 452 DLL3-14- VH
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGLCLEWMGIINPSEGS- TSYAQ
G44C- KFQGRVTMTRDTSTNTVYMDLSSLRSEDTAVYYCARGGNSAFYSYYDMDVWGQGTTVTVSS
D55E 453 DLL3-14- VH
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGLCLEWMGIINPSDAS-
TSYAQ G44C-
KFQGRVTMTRDTSTNTVYMDLSSLRSEDTAVYYCARGGNSAFYSYYDMDVWGQGTTVTVSS G56A
454 DLL3-14- VH
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGQCLEWMGIINPSEGS- TSYAQ
L43Q- KFQGRVTMTRDTSTNTVYMDLSSLRSEDTAVYYCARGGNSAFYSYYDMDVWGQGTTVTVSS
G44C- D55E 455 DLL3-14- VH
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGQCLEWMGIINPSDAS- TSYAQ
L43Q- KFQGRVTMTRDTSTNTVYMDLSSLRSEDTAVYYCARGGNSAFYSYYDMDVWGQGTTVTVSS
G44C- G56A 456 DLL3-14- VL
DVVMTQTPLSLPVTLGQPASISCRSSQSLVYREGNTYLSWFQQRPGQSPRRLIYKVS- NWQSG
D171E VPDRFSGGGSGTDFTLKISRVEAEDVGVYYCMQGTHWPPTFGQGTKVEIK 457
DLL3-14- VL
DVVMTQTPLSLPVTLGQPASISCRSSQSLVYRDANTYLSWFQQRPGQSPRRLIYKVS- NWQSG
G172A VPDRFSGGGSGTDFTLKISRVEAEDVGVYYCMQGTHWPPTFGQGTKVEIK 458
DLL3-14- VL
DVVMTQTPLSLPVTLGQPASISCRSSQSLVYRDGQTYLSWFQQRPGQSPRRLIYKVS- NWQSG
N173Q VPDRFSGGGSGTDFTLKISRVEAEDVGVYYCMQGTHWPPTFGQGTKVEIK 459
DLL3-14- VL
DVVMTQTPLSLPVTLGQPASISCRSSQSLVYRDGNAYLSWFQQRPGQSPRRLIYKVS- NWQSG
T174A VPDRFSGGGSGTDFTLKISRVEAEDVGVYYCMQGTHWPPTFGQGTKVEIK 460
DLL3-14- VL
DVVMTQTPLSLPVTLGQPASISCRSSQSLVYRDGNTYLSWFQQRPGQSPRRLIYKVS- NWQSG
G208S VPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPPTFGQGTKVEIK 461
DLL3-14- VL
DVVMTQTPLSLPVTLGQPASISCRSSQSLVYREGNTYLSWFQQRPGQSPRRLIYKVS- NWQSG
D171E- VPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPPTFGQGTKVEIK G208S VL
462 DLL3-14
DVVMTQTPLSLPVTLGQPASISCRSSQSLVYRDANTYLSWFQQRPGQSPRRLIYKVSNWQ- SG
G172A- VPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPPTFGQGTKVEIK G208S 463
DLL3-14- VL
DVVMTQTPLSLPVTLGQPASISCRSSQSLVYRDGNTYLSWFQQRPGQSPRRLIYKVS- NWQSG
Q243C VPDRFSGGGSGTDFTLKISRVEAEDVGVYYCMQGTHWPPTFGCGTKVEIK 464
DLL3-14- VL
DVVMTQTPLSLPVTLGQPASISCRSSQSLVYREGNTYLSWFQQRPGQSPRRLIYKVS- NWQSG
D171E- VPDRFSGGGSGTDFTLKISRVEAEDVGVYYCMQGTHWPPTFGCGTKVEIK Q243C 465
DLL3-14- VL
DVVMTQTPLSLPVTLGQPASISCRSSQSLVYRDANTYLSWFQQRPGQSPRRLIYKVS- NWQSG
G172A- VPDRFSGGGSGTDFTLKISRVEAEDVGVYYCMQGTHWPPTFGCGTKVEIK Q243C 466
DLL3-14- VL
DVVMTQTPLSLPVTLGQPASISCRSSQSLVYRDGQTYLSWFQQRPGQSPRRLIYKVS- NWQSG
N173Q VPDRFSGGGSGTDFTLKISRVEAEDVGVYYCMQGTHWPPTFGCGTKVEIK Q243C 467
DLL3-14- VL
DVVMTQTPLSLPVTLGQPASISCRSSQSLVYRDGNAYLSWFQQRPGQSPRRLIYKVS- NWQSG
T174A- VPDRFSGGGSGTDFTLKISRVEAEDVGVYYCMQGTHWPPTFGCGTKVEIK Q243C 468
DLL3-14- VL
DVVMTQTPLSLPVTLGQPASISCRSSQSLVYRDGNTYLSWFQQRPGQSPRRLIYKVS- NWQSG
G208S- VPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPPTFGCGTKVEIK Q243C 469
DLL3-14- VL
DVVMTQTPLSLPVTLGQPASISCRSSQSLVYREGNTYLSWFQQRPGQSPRRLIYKVS- NWQSG
D171E- VPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPPTFGCGTKVEIK G208S-
Q243C 470 DLL3-14- VL
DVVMTQTPLSLPVTLGQPASISCRSSQSLVYRDANTYLSWFQQRPGQSPRRLIYKVS- NWQSG
G172A- VPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPPTFGCGTKVEIK G208S-
Q243C 471 DLL3-14- scFv
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGLGLEWMGIINPSD- GSTSYAQ
001 KFQGRVTMTRDTSTNTVYMDLSSLRSEDTAVYYCARGGNSAFYSYYDMDVWGQGTTVTVSSG
GGGSGGGGSGGGGSDVVMTQTPLSLPVTLGQPASISCRSSQSLVYREGNTYLSWFQQRPGQS
PRRLIYKVSNWQSGVPDRFSGGGSGTDFTLKISRVEAEDVGVYYCMQGTHWPPTFGQGTKVE IK
472 DLL3-14- scFv
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGLGLEWMGIINPSD- GSTSYAQ
002 KFQGRVTMTRDTSTNTVYMDLSSLRSEDTAVYYCARGGNSAFYSYYDMDVWGQGTTVTVSSG
GGGSGGGGSGGGGSDVVMTQTPLSLPVTLGQPASISCRSSQSLVYRDANTYLSWFQQRPGQS
PRRLIYKVSNWQSGVPDRFSGGGSGTDFTLKISRVEAEDVGVYYCMQGTHWPPTFGQGTKVE IK
473 DLL3-14- scFv
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGLGLEWMGIINPSD- GSTSYAQ
003 KFQGRVTMTRDTSTNTVYMDLSSLRSEDTAVYYCARGGNSAFYSYYDMDVWGQGTTVTVSSG
GGGSGGGGSGGGGSDVVMTQTPLSLPVTLGQPASISCRSSQSLVYRDGQTYLSWFQQRPGQS
PRRLIYKVSNWQSGVPDRFSGGGSGTDFTLKISRVEAEDVGVYYCMQGTHWPPTFGQGTKVE IK
474 DLL3-14- scFv
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGLGLEWMGIINPSD- GSTSYAQ
004 KFQGRVTMTRDTSTNTVYMDLSSLRSEDTAVYYCARGGNSAFYSYYDMDVWGQGTTVTVSSG
GGGSGGGGSGGGGSDVVMTQTPLSLPVTLGQPASISCRSSQSLVYRDGNAYLSWFQQRPGQS
PRRLIYKVSNWQSGVPDRFSGGGSGTDFTLKISRVEAEDVGVYYCMQGTHWPPTFGQGTKVE IK
475 DLL3-14- scFv
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGLGLEWMGIINPSD- GSTSYAQ
005 KFQGRVTMTRDTSTNTVYMDLSSLRSEDTAVYYCARGGNSAFYSYYDMDVWGQGTTVTVSSG
GGGSGGGGSGGGGSDVVMTQTPLSLPVTLGQPASISCRSSQSLVYRDGNTYLSWFQQRPGQS
PRRLIYKVSNWQSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPPTFGQGTKVE IK
476 DLL3-14- scFv QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGQG